Combination therapy of antibodies against human csf-1r and tlr9 agonist

ABSTRACT

The present invention relates to the combination therapy of antibodies against human CSF-1R with a TLR9 agonist.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/257,480filed on Apr. 21, 2014, which claims the priority benefit of EuropeanPatent Application No. 13164695.2 filed Apr. 22, 2013, the disclosure ofeach of which is hereby incorporated by reference in its entirety forall purpose.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:146392030010SeqList.TXT,date recorded: Oct. 15, 2015, size: 88 KB).

FIELD OF THE INVENTION

The present invention relates to the combination therapy comprisingantibodies against human CSF-1R and a TLR9 agonist.

BACKGROUND OF THE INVENTION

CSF-1R and CSF-1R antibodies

The human CSF-1 receptor (CSF-1R; colony stimulating factor 1 receptor;synonyms: M-CSF receptor; Macrophage colony-stimulating factor 1receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 62) is known since 1986(Coussens, L., et al., Nature 320 (1986) 277-280). CSF-1R is a growthfactor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth,P., and Stanley, E. R., Curr. Top. Microbiol. Immunol. 181 (1992)141-167).

CSF-1R is the receptor for CSF-1 (colony stimulating factor 1, alsocalled M-CSF, macrophage colony-stimulating factor) and mediates thebiological effects of this cytokine (Sherr, C. J., et al., Cell 41(1985) 665-676). The cloning of the colony stimulating factor-1 receptor(CSF-1 R) (also called c-fms) was described for the first time inRoussel, M. F., et al., Nature 325 (1987) 549-552. In that publication,it was shown that CSF-1R had transforming potential dependent on changesin the C-terminal tail of the protein including the loss of theinhibitory tyrosine 969 phosphorylation which binds Cb1 and therebyregulates receptor down regulation (Lee, P. S., et al., Embo J. 18(1999) 3616-3628). Recently a second ligand for CSF-1R termedinterleukin-34 (IL-34) was identified (Lin, H., et al, Science 320(2008) 807-811).

Currently two CSF-1R ligands that bind to the extracellular domain ofCSF-1R are known. The first one is CSF-1 (colony stimulating factor 1,also called M-CSF, macrophage; SEQ ID NO: 86) and is foundextracellularly as a disulfide-linked homodimer (Stanley, E. R. et al.,Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, E. R. etal., Stem Cells 12 Suppl. 1 (1995) 15-24). The second one is IL-34(Human IL-34; SEQ ID NO: 87) (Hume, D. A., et al, Blood 119 (2012)1810-1820). The main biological effects of CSF-1R signaling are thedifferentiation, proliferation, migration, and survival of hematopoieticprecursor cells to the macrophage lineage (including osteoclast).Activation of CSF-1R is mediated by its CSF-1R ligands, CSF-1 (M-CSF)and IL-34. Binding of CSF-1 (M-CSF) to CSF-1R induces the formation ofhomodimers and activation of the kinase by tyrosine phosphorylation (Li,W. et al, EMBO Journal. 10 (1991) 277-288; Stanley, E. R., et al., Mol.Reprod. Dev. 46 (1997) 4-10).

The biologically active homodimer CSF-1 binds to the CSF-1R within thesubdomains D1 to D3 of the extracellular domain of the CSF-1 receptor(CSF-1R-ECD). The CSF-1R-ECD comprises five immunoglobulin-likesubdomains (designated D1 to D5). The subdomains D4 to D5 of theextracellular domain (CSF-1R-ECD) are not involved in the CSF-1 binding(Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359).The subdomain D4 is involved in dimerization (Yeung, Y-G., et alMolecular & Cellular Proteomics 2 (2003) 1143-1155; Pixley, F. J., etal., Trends Cell Biol 14 (2004) 628-638).

Further signaling is mediated by the p85 subunit of PI3K and Grb2connecting to the PI3K/AKT and Ras/MAPK pathways, respectively. Thesetwo important signaling pathways can regulate proliferation, survivaland apoptosis. Other signaling molecules that bind the phosphorylatedintracellular domain of CSF-1R include STAT1, STAT3, PLCy, and Cb1(Bourette, R. P. and Rohrschneider, L. R., Growth Factors 17 (2000)155-166).

CSF-1R signaling has a physiological role in immune responses, in boneremodeling and in the reproductive system. The knockout animals foreither CSF-1 (Pollard, J. W., Mol. Reprod. Dev. 46 (1997) 54-61) orCSF-1R (Dai, X. M., et al., Blood 99 (2002) 111-120) have been shown tohave osteopetrotic, hematopoietic, tissue macrophage, and reproductivephenotypes consistent with a role for CSF-1R in the respective celltypes.

Sherr, C. J., et al., Blood 73 (1989) 1786-1793 relates to someantibodies against CSF-1R that inhibit the CSF-1 activity. Ashmun, R.A., et al., Blood 73 (1989) 827-837 relates to CSF-1R antibodies. Lenda,D., et al., Journal of Immunology 170 (2003) 3254-3262 relates toreduced macrophage recruitment, proliferation, and activation inCSF-1-deficient mice results in decreased tubular apoptosis during renalinflammation. Kitaura, H., et al., Journal of Dental Research 87 (2008)396-400 refers to an anti-CSF-1 antibody which inhibits orthodontictooth movement. WO 2001/030381 mentions CSF-1 activity inhibitorsincluding antisense nucleotides and antibodies while disclosing onlyCSF-1 antisense nucleotides. WO 2004/045532 relates to metastases andbone loss prevention and treatment of metastatic cancer by a CSF-1antagonist disclosing as antagonist anti-CSF-1-antibodies only. WO2005/046657 relates to the treatment of inflammatory bowel disease byanti-CSF-1-antibodies. US 2002/0141994 relates to inhibitors of colonystimulating factors. WO 2006/096489 relates to the treatment ofrheumatoid arthritis by anti-CSF-1-antibodies. WO 2009/026303 and WO2009/112245 relate to certain anti-CSF-1R antibodies binding to CSF-1Rwithin the first three subdomains (D1 to D3) of the Extracellular Domain(CSF-1R-ECD). WO2011/123381(A1) relates to antibodies against CSF-1R.WO2011/070024 relate to certain anti-CSF-1R antibodies binding to CSF-1Rwithin the dimerization domain (D4 to D5).

TLRs, TLR9 and TLR9 Agonists

Different experimental Toll-like receptor agonists for cancer therapyare described (Galluzzi et al., Oncolmmunology, 1:5, (2012) 699-716)Toll-like receptors (TLRs) in general are prototypic pattern recognitionreceptors (PRRs) best known for their ability to activate the innateimmune system in response to conserved microbial components such aslipopolysaccharide and double-stranded RNA. Accumulating evidenceindicates that the function of TLRs is not restricted to the elicitationof innate immune responses against invading pathogens. TLRs have indeedbeen shown to participate in tissue repair and injury-inducedregeneration as well as in adaptive immune responses against cancer. Inparticular, TLR4 signaling appears to be required for the efficientprocessing and cross-presentation of cell-associated tumor antigens bydendritic cells, which de facto underlie optimal therapeutic responsesto some anticancer drugs. Thus, TLRs constitute prominent therapeutictargets for the activation/intensification of anticancer immuneresponses. In line with this notion, long-used preparations such as theColey toxin (a mixture of killed Streptococcus pyogenes and Serratiamarcescens bacteria) and the bacillus Calmette-Guérin (BCG, anattenuated strain of Mycobacterium bovis originally developed as avaccine against tuberculosis), both of which have been associated withconsistent anticancer responses, potently activate TLR2 and TLR4signaling.

According to currently accepted models, TLRs operate as homo- orhetero-dimers and are expressed either at the plasma membrane (TLRs thatmainly bind proteo-lipidic MAMPs, i.e., TLR1, TLR2, TLR4, TLR5, TLR6 andTLR10) or in endosomes (TLRs that detect microbial nucleic acids, i.e.,TLR3, TLR7, TLR8, TLR9). TLR10, which is the only orphan receptor amonghuman TLRs, has also been shown to co-localize with TLR2 at phagosomes,suggesting that it may share with TLR2 the ability to bind acylatedlipopeptides. Conclusive data on this issue, however, have not yet beenreported. TLR11-13 are not encoded in the human genome. In mice,TLR11-13 are constitutively expressed in the central nervous system andundergo several-fold induction in response to cysticercosis.21 TLR11reportedly recognizes a profilin-like protein expressed by Toxoplasmagondii and has been localized at the endoplasmic reticulum. TLR13 alsoappears to be localized intracellularly, where it would specificallydetect the vesicular stomatitis virus. So far, the ligand specificityand intracellular localization of TLR12 remain unexplored.

So in summary the different Toll-like receptors have differentfunctions, structure and expression patterns. Consequently also theirligands and agonist have different functions and mode of action. E.g.LPS, the natural ligand of TLR2 and TLR4 also known as endotoxin, hasanticancer properties which have been discovered as early as in the1960s, when the existence of TLRs was not even suspected.

TLR9 is mainly found in the endosomal compartment of B cells, monocytes,macrophages and plasmacytoid Dendritic Cells DCs (Galluzzi et al.,Oncolmmunology, 1:5, (2012) 699-716). The main ligand of TLR9 isbacterial/viral DNA, differing from its mammalian counterpart for theelevated frequency of unmethylated CpG oligodeoxynucleotides. Indeed,whereas mammalian DNA has no immunostimulatory activity, theadministration of bacterial/viral DNA induces a potent Th1 immuneresponse in vivo, which is entirely abrogated in TLR9^(−/−) mice. CpGoligodeoxynucleotides (or CpG ODN) are short single-stranded syntheticDNA molecules that contain a cytidine triphosphate deoxynucleotide (“C)”followed by a guanidine triphosphate deoxynucleotide (“G”). The “p”refers to the phosphodiester link between consecutive nucleotides,although some ODN have a modified phosphorothioate (PS) backboneinstead. When these CpG motifs are unmethlyated, they act asimmunostimulants (Weiner, G J; et al, PNAS 94 (1997) 10833-7).

CpG motifs are considered pathogen-associated molecular patterns (PAMPs)due to their abundance in microbial genomes but their rarity invertebrate genomes (Bauer, S; Current Topics in Microbiology andImmunology 270 (2002) 145-54). The CpG PAMP is recognized by the patternrecognition receptor (PRR) Toll-Like Receptor 9 (TLR9), which isconstitutively expressed only in B cells and plasmacytoid dendriticcells (pDCs) in humans and other higher primates (Rothenfusser, S; etal, Human immunology 63 (2002) 1111-9)

Synthetic CpG ODN differ from microbial DNA in that they have apartially or completely phosphorothioated (PS) backbone instead of thetypical phosphodiester backbone and a poly G tail at the 3′ end, 5′ end,or both. PS modification protects the ODN from being degraded bynucleases such as DNase in the body and poly G tail enhances cellularuptake (Dalpke, A H et al, Immunology 106 (2002) 102-12). The poly Gtails form intermolecular tetrads that result in high molecular weightaggregates. These aggregates are responsible for the increased activitythe poly G sequence impart; not the sequence itself

These synthetic oligodeoxynucleotides containing unmethylated CpG motifs(CpG ODNs), such as ODN 1826, have been extensively studied as adjuvants(Steinhagen F. et al., 2011; Vaccine 29(17):3341-55). These CpG motifsare present at a 20-fold greater frequency in bacterial DNA compared tomammalian DNA (Hemmi H. et al., 2000. Nature 408: 740-5). CpG ODNsagonize TLR9, which is expressed on human B cells and plasmacytoiddendritic cells (pDCs), thereby inducing Th1-dominated immune responses(Coffman et al., 2010. Immunity 33(4):492-503). Pre-clinical studies,conducted in rodents and non-human primates, and human clinical trialshave demonstrated that CpG ODNs can significantly improvevaccine-specific antibody responses (Steinhagen F. et al., 2011; Vaccine29(17):3341-55).

Numerous sequences have been shown to stimulate TLR9 with variations inthe number and location of CpG dimers, as well as the precise basesequences flanking the CpG dimers. This led to the creation of classesor categories of CpG ODN, which are all TLR9 agonist based on theirsequence, secondary structures, and effect on human peripheral bloodmononuclear cells (PBMCs). The three main classes of CpG ODNs are classA, B and C, which differ in their immune-stimulatory activities (Krug A.et al., 2001, Eur J Immunol, 31(7): 2154-63). Furthermore, CpG ODNsactivate TLR9 in a species-specific manner (Bauer, S. et al., 2001,PNAS, 98(16):9237-42). One of the first Class A ODN, ODN 2216, wasdescribed in 2001 by Krug et al (see above) This class of ODN wasdistinctly different from the previously described Class B ODN (i.e.,ODN 2006) in that it stimulated the production of large amounts of TypeI interferons, the most important one being IFNα, and induced thematuration of pDCs.

Class A ODN are also strong activators of NK cells through indirectcytokine signaling. Class A ODN typically contain 7 to 10 PS-modifiedbases at one or both ends that resist degradation by nucleases andincrease the longevity of the ODN. The above rules strictly define theclass, but variability of the sequence within these “rules” is possible.It should also be noted that changes to the sequence will affect themagnitude of the response. For example, the internal palindrome sequencecan be 4 to 8 base pairs in length and vary in the order of bases,however the pattern, 5′-Pu Pu CG Pu Py CG Py Py-3′, was found to be themost active when compared to several other sequences. The poly G tailfound at either end of the DNA strand can vary in length and evennumber, but its presence is critical to the activity of the molecule.

Class B ODN (i.e. ODN 2007) are strong stimulators of human B cell andmonocyte maturation. They also stimulate the maturation of pDC but to alesser extent than Class A ODN and very small amounts of IFNα. Thestrongest ODN in this class have three 6 mer sequences. Class B ODNshave been studied extensively as therapeutic agents because of theirability to induce a strong humoral immune response, making them ideal asa vaccine adjuvant.

ODN 1826 is a type B CpG ODN specific for mouse TLR9. Type B CpG ODNscontain a full phosphorothioate backbone with one or more CpGdinucleotides and can strongly activate B cells (Krug A. et al., 2001,Eur J Immunol, 31(7): 2154-63). ODN 1826, a mouse-reactive surrogateTLR9-agonist has been tested as an adjuvant in numerous animal models(Bauer, S. et al., 2001, PNAS, 98(16):9237-42). Research in micedemonstrated that ODN 1826 administration can induce the activation ofantigen presenting cells and type I IFN anti-viral activity 8-9,indicative of a Th1 immune response (Longhi Mp. et al., 2009, J Exp Med206: 1589-602).

Moreover, the administration of type B CpG oligonucleotides (alone orcombined with chemotherapeutics or peptide vaccines) to tumor-bearingrodents reportedly exerts potent anticancer effects. Initial Phase I/IIclinical trials to test the safety and efficacy of CpG-7909 foroncological indications were launched in April 2000. Approximately inthe same period, CpG-7909 begun to be extensively investigated as anadjuvant for cancer-unrelated indications (mainly antiviral vaccines),showing no severe side effects and encouraging efficacy.

During the last decade, the safety and anticancer potential of CpG-7909(as a standalone agent or in combination with chemotherapy and/orvaccination approaches) have been investigated in a large number ofPhase I/II clinical trials, including studies with leukemia, lymphoma,basal cell carcinoma, lmelanoma, esophageal squamous cell carcinoma,NSCLC, renal cell carcinoma, and prostate cancer patients. Several TLR9agonist are known and currently developed in clinical testing Agatolimod(tricosasodium salt of a synthetic 24-mer oligonucleotide containing 3CpG motifs; Pfizer) GNKG168 (CpG ODN; SBI Biotech), IMO-2055 (syntheticoligonucleotide containing unmethylated CpG dinucleotides; IderaPharmaceuticals), MGN-1703 (Mologen). Typically these TLR9 agonist areused in the treatment of different cancers:

Schroder K et al, (J Leukoc Biol. 81(6) (2007) 1577-90 relates to TLRagonist (unmethylated CpG-containing DNA (CpG DNA)) the regulation ofmouse T1R9 expression and defines a molecular mechanism by whichIFN-gamma amplifies mouse macrophage responses to CpG DNA.

SUMMARY OF THE INVENTION

The invention comprises the combination therapy of an antibody bindingto human CSF-1R (including antibodies binding to domains D1-D3 andantibodies binding to domains D4-D5, preferably antibodies binding todomains D4-D5 as described herein) with a Toll-like receptor 9 (TLR9)agonist for use in the treatment of cancer.

One embodiment is an antibody which binds to human CSF-1R characterizedin binding to the (dimerization) domains D4 to D5 (SEQ ID No: 85) of theextracellular domain of human CSF-1R for use in

-   -   a) the inhibition of cell proliferation in CSF-1R        ligand-dependent and/or CSF-1 ligand-independent CSF-1R        expressing tumor cells;    -   b) the inhibition of cell proliferation of tumors with CSF-1R        ligand-dependent and/or CSF-1R ligand-independent CSF-1R        expressing macrophage infiltrate;    -   c) the inhibition of cell survival (in CSF-1R ligand-dependant        and/or CSF-1R ligand-independent) CSF-1R expressing monocytes        and macrophages; and/or    -   d) the inhibition of cell differentiation (in CSF-1R        ligand-dependent and/or CSF-1R ligand-independent) CSF-1R        expressing monocytes into macrophages, wherein the anti-CSF-1R        antibody is administered in combination with TLR9 agonist.

In one embodiment the TLR9 agonist is characterized by induction ofIFN-alpha, IL-6, and/or IL-12 (elevating the levels of IFN-alpha, IL-6,and/or IL-12) in plasmacytoid dendritic cells (pDCs). In one embodimentthe TLR9 agonist is characterized by elevating the level of IFN-alpha inhuman plasmacytoid dendritic cells (pDCs) (as measured by sandwichELISA).

In one embodiment of the invention the TLR9 agonist of the invention isa oligodeoxynucleotides containing a) cytosine-phosphate-guanosine (CpG)motifs (CpG ODNs) b) pyrimidine-phosphate-guanosine (YpG) motifs (YpGODNs) or c) cytosine-phosphate-purine (CpR) motifs (CpR ODNs).

In one embodiment of the invention the TLR9 agonist of the invention isa oligodeoxynucleotides containing cytosine-phosphate-guanosine (CpG)motifs (CpG ODNs).

CSF-1R antibodies binding to domains D1-D3 of human CSF-1R are describede.g. in WO 2009/026303 and WO 2009/112245 relate to certain anti-CSF-1Rantibodies binding to CSF-1R within the first three subdomains (D1 toD3) of the Extracellular Domain (CSF-1R-ECD). WO2011/123381(A1) relatesto antibodies against CSF-1R. and Sherr, C. J., et al., Blood 73 (1989)1786-1793 (typically these antibodies are characterized by inhibitingCSF-1R ligand-dependent but not CSF-1R ligand-independent CSF-1Rproliferation and/or signaling).

CSF-1R antibodies binding to domains D4-D5 of human CSF-1R are describede.g. within the present invention, in WO2011/070024, inPCT/EP2012/075241 and Sherr, C. J., et al., Blood 73 (1989) 1786-1793(typically these antibodies are characterized by inhibiting CSF-1Rligand-dependent and CSF-1R ligand-independent CSF-1R proliferationand/or signaling).

In one embodiment is an antibody which binds to human CSF-1R ischaracterized in binding to the (dimerization) domains D4 to D5 (SEQ IDNo: 85) of the extracellular domain of human CSF-1R.

In one embodiment of the invention the anti-CSF-1R antibody ischaracterized in that the antibody binds to human CSF-1R fragment delD4(SEQ ID NO: 65) and to human CSF-1R Extracellular Domain (SEQ ID NO: 64)with a ratio of 1:50 or lower.

In one embodiment of the invention the antibody is characterized in thatthe antibody does not bind to human CSF-1R fragment delD4 (SEQ ID NO:65).

In one embodiment of the invention the antibody is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:7 and the light        chain variable domain is SEQ ID NO:8,    -   b) the heavy chain variable domain is SEQ ID NO:15 and the light        chain variable domain is SEQ ID NO:16;    -   c) the heavy chain variable domain is SEQ ID NO:75 and the light        chain variable domain is SEQ ID NO:76;    -   d) the heavy chain variable domain is SEQ ID NO:83 and the light        chain variable domain is SEQ ID NO:84;        or a humanized version thereof.

In one embodiment of the invention the antibody is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:7 and the light        chain variable domain is SEQ ID NO:8,    -   b) the heavy chain variable domain is SEQ ID NO:15 and the light        chain variable domain is SEQ ID NO:16;        or a humanized version thereof

In one embodiment of the invention the antibody is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24, or    -   b) the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32, or    -   c) the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40, or    -   d) the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48, or    -   e) the heavy chain variable domain is SEQ ID NO:55 and the light        chain variable domain is SEQ ID NO:56.

In one embodiment of the invention the antibody is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region        of SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54; or    -   h) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region        of SEQ ID NO:71, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO:73, and        a CDR1 region of SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region        of SEQ ID NO: 79, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:80, a CDR2 region of SEQ ID NO: 81,        and a CDR1 region of SEQ ID NO: 82.

One embodiment of the invention is an antibody binding to human CSF-1R,for use in the treatment of a patient having a CSF-1R expressing tumoror having a tumor with CSF-1R expressing macrophage infiltrate, whereinthe tumor is characterized by an increase of CSF-1R ligand and whereinthe anti-CSF-1R antibody is administered in combination with a TLR9agonist.

In one embodiment of the invention the antibody is of human IgG1subclass or of human IgG4 subclass.

The invention further comprises the use an of an CSF-1R antibodyaccording to the invention for the manufacture of a medicament fortreatment of a CSF-1R mediated disease in combination with a TLR9agonist.

The invention further comprises the use an of an CSF-1R antibodyaccording to the invention for the manufacture of a medicament fortreatment of cancer in combination with a TLR9 agonist.

The invention further comprises the use an of an CSF-1R antibodyaccording to the invention for the manufacture of a medicament fortreatment of bone loss in combination with a TLR9 agonist.

The invention further comprises the use an of an CSF-1R antibodyaccording to the invention for the manufacture of a medicament fortreatment of metastasis in combination with a TLR9 agonist.

The invention further comprises the use an of an CSF-1R antibodyaccording to the invention for the manufacture of a medicament fortreatment of inflammatory diseases in combination with a TLR9 agonist.

The invention further comprises an CSF-1R antibody according to theinvention for treatment of a CSF-1R mediated disease in combination witha TLR9 agonist.

The invention further comprises an CSF-1R antibody according to theinvention for treatment of cancer in combination with a TLR9 agonist.

The invention further comprises an CSF-1R antibody according to theinvention for treatment of bone loss in combination with a TLR9 agonist.

The invention further comprises an CSF-1R antibody according to theinvention for treatment of metastasis in combination with a TLR9agonist.

The invention further comprises an CSF-1R antibody according to theinvention for treatment of inflammatory diseases in combination with aTLR9 agonist.

The combination therapies of the antibodies described herein showbenefits for patients in need of a CSF-1R targeting therapy.

The antibodies according to the invention show efficientantiproliferative activity against ligand-independent andligand-dependent proliferation and are therefore especially useful inthe treatment of cancer and metastasis in combination with a TLR9agonist.

The invention further provides a method for treating a patient sufferingfrom cancer, comprising administering to a patient diagnosed as havingsuch a disease (and therefore being in need of such a therapy) aneffective amount of an CSF-1R antibody according to the invention incombination with a TLR-9 agonist.

The invention also provides compositions comprising an antibody whichbinds to human CSF-1R and a Toll-like receptor 9 (TLR9) agonist. In someembodiments, the antibody does not bind to human CSF-1R fragment delD4(SEQ ID NO: 65). In some embodiments, the antibody comprises a) a heavychain variable domain comprising SEQ ID NO:7 and a light chain variabledomain comprising SEQ ID NO:8, b) a heavy chain variable domaincomprising SEQ ID NO:15 and a light chain variable domain comprises SEQID NO:16; c) a heavy chain variable domain comprising SEQ ID NO:75 and alight chain variable domain comprising SEQ ID NO:76; d) a heavy chainvariable domain comprising SEQ ID NO:83 and a light chain variabledomain comprising SEQ ID NO:84; e) a heavy chain variable domaincomprising SEQ ID NO:23 and a light chain variable domain comprising SEQID NO:24, or f) a heavy chain variable domain comprising SEQ ID NO:31and the light chain variable domain comprising SEQ ID NO:32, or g) aheavy chain variable domain comprising SEQ ID NO:39 and the light chainvariable domain comprising SEQ ID NO:40, or h) a heavy chain variabledomain comprising SEQ ID NO:47 and the light chain variable domaincomprising SEQ ID NO:48, or i) a heavy chain variable domain comprisingSEQ ID NO:55 and a light chain variable domain comprising SEQ ID NO:56.In some embodiments, the antibody comprises a) a heavy chain variabledomain comprising a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ IDNO: 2, and a CDR1 region of SEQ ID NO:3, and a light chain variabledomain comprising a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ IDNO:5, and a CDR1 region of SEQ ID NO:6, or b) a heavy chain variabledomain comprising a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ IDNO: 10, and a CDR1 region of SEQ ID NO: 11, and a light chain variabledomain comprising a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ IDNO: 13, and a CDR1 region of SEQ ID NO: 14, or c) a heavy chain variabledomain comprising a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQID NO: 18, and a CDR1 region of SEQ ID NO:19, and light chain variabledomain comprising a CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQID NO:21, and a CDR1 region of SEQ ID NO:22, or d) a heavy chainvariable domain comprising a CDR3 region of SEQ ID NO: 25, a CDR2 regionof SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and a light chainvariable domain comprising a CDR3 region of SEQ ID NO:28, a CDR2 regionof SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or e) a heavychain variable domain comprising a CDR3 region of SEQ ID NO: 33, a CDR2region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and a lightchain variable domain comprising a CDR3 region of SEQ ID NO:36, a CDR2region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or f) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO:41, aCDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and alight chain variable domain comprising a CDR3 region of SEQ ID NO: 44, aCDR2 region of SEQ ID NO:45, and a CDR1 region of SEQ ID NO:46, or g) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO: 49, aCDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and alight chain variable domain comprising a CDR3 region of SEQ ID NO:52, aCDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54; or h)a heavy chain variable domain comprising a CDR3 region of SEQ ID NO:69,a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO:71, and alight chain variable domain comprising a CDR3 region of SEQ ID NO: 72, aCDR2 region of SEQ ID NO:73, and a CDR1 region of SEQ ID NO:74, or i) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO: 77, aCDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and alight chain variable domain comprising a CDR3 region of SEQ ID NO:80, aCDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82.

Even another embodiment of the invention provides methods of treatingcancer. The methods comprise administering an effective amount of i) anantibody which binds to human CSF-1R, and ii) TLR9 agonist. In someembodiments, the the cancer expresses or overexpresses CSF-1R. in someembodiments, the cancer is breast cancer, colorectal cancer, melanoma,head and neck cancer, lung cancer or prostate cancer.

Yet another embodiment of the invention provides methods of treatingcancer. The methods comprise administering an effective amount of anantibody which specifically binds to the dimerization domains D4 to D5(SEQ ID No: 85) of the extracellular domain of human CSF-1R and a TLR9agonist, wherein a) cell proliferation in CSF-1R ligand-dependent and/orCSF-1 ligand-independent CSF-1R expressing tumor cells is inhibited; b)cell proliferation of tumors with CSF-1R ligand-dependent and/or CSF-1Rligand-independent CSF-1R expressing macrophage infiltrate is inhibited;c) cell survival (in CSF-1R ligand-dependant and/or CSF-1Rligand-independent) CSF-1R expressing monocytes and macrophages isinhibited; or d) cell differentiation (in CSF-1R ligand-dependent and/orCSF-1R ligand-independent) CSF-1R expressing monocytes into macrophagesis inhibited.

Another embodiment of the invention provides methods of treating apatient having a CSF-1R expressing tumor or having a tumor with CSF-1Rexpressing macrophage infiltrate, wherein the tumor expresses increasedlevels of CSF-1R ligand. The method comprising administering aneffective amount of an antibody which specifically binds to human CSF-1Rand w a TLR9 agonist. In some embodiments, the TLR9 agonist inducesIFN-alpha, IL-6, and/or IL-12 in plasmacytoid dendritic cells (pDCs). Insome embodiments, the TLR9 agonist is an oligodeoxynucleotide containingcytosine-phosphate-guanosine (CpG) motifs (CpG ODNs). In someembodiments, the antibody specifically binds to the domains D4 to D5(SEQ ID No: 85) of the extracellular domain of human CSF-1R. In someembodiments, the antibody does not bind to human CSF-1R fragment delD4(SEQ ID NO: 65). In some embodiments, the antibody comprises a) a heavychain variable domain comprising SEQ ID NO:7 and a light chain variabledomain comprising SEQ ID NO:8, b) a heavy chain variable domaincomprising SEQ ID NO:15 and a light chain variable domain comprising SEQID NO:16; c) a heavy chain variable domain comprising SEQ ID NO:75 and alight chain variable domain comprising SEQ ID NO:76; d) a heavy chainvariable domain comprising SEQ ID NO:83 and a light chain variabledomain comprising SEQ ID NO:84; e) a heavy chain variable domaincomprising SEQ ID NO:23 and a light chain variable domain comprising SEQID NO:24, or f) a heavy chain variable domain comprising SEQ ID NO:31and a light chain variable domain comprising SEQ ID NO:32, or g) a heavychain variable domain comprising SEQ ID NO:39 and a light chain variabledomain comprising SEQ ID NO:40, or h) a heavy chain variable domaincomprising SEQ ID NO:47 and a light chain variable domain comprising SEQID NO:48, or i) a heavy chain variable domain comprising SEQ ID NO:55and a light chain variable domain comprising SEQ ID NO:56. In someembodiments, the antibody comprises a) a heavy chain variable domaincomprising a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2,and a CDR1 region of SEQ ID NO:3, and a light chain variable domaincomprising a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5,and a CDR1 region of SEQ ID NO:6, or b) a heavy chain variable domaincomprising a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO:10, and a CDR1 region of SEQ ID NO: 11, and a light chain variabledomain comprising a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ IDNO: 13, and a CDR1 region of SEQ ID NO: 14, or c) a heavy chain variabledomain comprising a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQID NO: 18, and a CDR1 region of SEQ ID NO:19, and a light chain variabledomain comprising a CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQID NO:21, and a CDR1 region of SEQ ID NO:22, or d) a heavy chainvariable domain comprising a CDR3 region of SEQ ID NO: 25, a CDR2 regionof SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and a light chainvariable domain comprising a CDR3 region of SEQ ID NO:28, a CDR2 regionof SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or e) a heavychain variable domain comprising a CDR3 region of SEQ ID NO: 33, a CDR2region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and a lightchain variable domain comprising a CDR3 region of SEQ ID NO:36, a CDR2region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or f) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO:41, aCDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and alight chain variable domain comprising a CDR3 region of SEQ ID NO: 44, aCDR2 region of SEQ ID NO:45, and a CDR1 region of SEQ ID NO:46, or g) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO: 49, aCDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and alight chain variable domain comprising a CDR3 region of SEQ ID NO:52, aCDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54; or h)a heavy chain variable domain comprising a CDR3 region of SEQ ID NO:69,a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO:71, and alight chain variable domain comprising a CDR3 region of SEQ ID NO: 72, aCDR2 region of SEQ ID NO:73, and a CDR1 region of SEQ ID NO:74, or i) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO: 77, aCDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and alight chain variable domain comprising a CDR3 region of SEQ ID NO:80, aCDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82. Insome embodiment, the antibody is human IgG1 subclass or human IgG4subclass.

Another embodiment of the invention provides methods of treating apatient having a CSF-1R expressing tumor or having a tumor with CSF-1Rexpressing macrophage infiltrate, wherein the tumor is characterized byan increase of CSF-1R ligand. The methods comprise administering aneffective amount of an an antibody which binds to human CSF-1R and aTLR9 agonist.

These and other embodiment of the invention will be described in greaterdetail in the detailed description that follows.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates data demonstrating the in vivo efficacy of acombination of a <CSF1R> antibody with a TLR9 agonist in the MC38 mouseCRC in vivo model—Median time to progression. Addition of TLR9 agonistto anti-CSF-1R antibody therapy resulted in a statistically significantmore than additive improvement of median time to progression (46 days)compared to anti-CSF-1R antibody monotherapy or TLR9 agonistmonotherapy.

FIG. 2A-2B illustrate data demonstrating growth inhibition of BeWo tumorcells in 3D culture under treatment with different anti-CSF-1Rmonoclonal antibodies at a concentration of 10 ug/ml.

X axis: viability normalized mean relative light units (RLU)corresponding to the ATP-content of the cells (CellTiterGlo® Assay).Y axis: tested probes: Minimal Medium (0.5% FBS), mouse IgG1 (mIgG1, 10μm/ml), mouse IgG2a (mIgG2a 10 μg/ml), CSF-1 only, Mab 2F11, Mab 2E10,Mab2H7, Mab1G10 and SC 2-4A5.Highest inhibition of CSF-1 induced growth was observed with theanti-CSF-1R antibodies according to the invention.

FIG. 3A-3F illustrates data demonstrating binding of differentanti-CSF-1R antibodies to immobilized human CSF-1R FIG. 3A: Biacoresensogram of binding of different anti-CSF-1R antibodies to immobilizedhuman CSF-1R fragment delD4 (comprising the extracellular subdomainsD1-D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal in Response Units(RU), baseline=0 RU, x-axis: time in seconds (s)): While the antibodiesMab 3291 and sc 2-4A5 clearly show binding to this delD4 fragment, theantibodies according to the invention e.g. Mab 2F11, and Mab 2E10, didnot bind to the CSF-1R fragment delD4. The control anti-CCR5 antibodym<CCR5>Pz03.1C5 did also not bind to the CSF-1R fragment delD4. FIG. 3B:Biacore sensogram of binding of different anti-CSF-1R antibodies toimmobilized human CSF-1R Extracellular Domain (CSF-1R-ECD) (comprisingthe extracellular subdomains D1-D5) (SEQ ID NO: 64) (y-axis: bindingsignal in Response Units (RU), baseline=0 RU, x-axis: time in seconds(s)): All anti-CSF-1R antibodies show binding to CSF-1R-ECD. The controlanti-CCR5 antibody m<CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD. FIG.3C: Biacore sensogram of binding of different anti-CSF-1R antibodies toimmobilized human CSF-1R fragment delD4 (comprising the extracellularsubdomains D1-D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal inResponse Units (RU), baseline=0 RU, x-axis: time in seconds (s)): Mab1G10, Mab 2H7 and humanized hMab 2F11-e7 did not bind to the CSF-1Rfragment delD4. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did alsonot bind to the CSF-1R fragment delD4. FIG. 3D: Biacore sensogram ofbinding of different anti-CSF-1R antibodies to immobilized human CSF-1RExtracellular Domain (CSF-1R-ECD) (comprising the extracellularsubdomains D1-D5) (SEQ ID NO: 64) (y-axis: binding signal in ResponseUnits (RU), baseline=0 RU, x-axis: time in seconds (s)): All anti-CSF-1Rantibodies Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 showed bindingto CSF-1R-ECD. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did notbind to the CSF-1R-ECD. FIG. 3E: Biacore sensogram of binding ofdifferent anti-CSF-1R antibodies to immobilized human CSF-1R fragmentdelD4 (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO:65) (y-axis: binding signal in Response Units (RU), baseline=0 RU,x-axis: time in seconds (s)): All anti-CSF-1R antibodies 1.2.SM, CXIIG6,ab10676 and MAB3291 show binding to the CSF-1R fragment delD4. Thecontrol anti-CCR5 antibody m<CCR5>Pz03.1C5 did also not bind to theCSF-1R fragment delD4. FIG. 3F: Biacore sensogram of binding ofdifferent anti-CSF-1R antibodies to immobilized human CSF-1RExtracellular Domain (CSF-1R-ECD) (comprising the extracellularsubdomains D1-D5) (SEQ ID NO: 64) (y-axis: binding signal in ResponseUnits (RU), baseline=0 RU, x-axis: time in seconds (s)): All anti-CSF-1Rantibodies 1.2.SM, CXIIG6, ab10676 and MAB3291 show binding toCSF-1R-ECD. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did not bindto the CSF-1R-ECD.

FIG. 4A-4D illustrates data showing CSF-1 levels in Cynomolgous monkeyafter administration of different dosages of anti-CSF-1R antibody. FIG.4A: CSF-1 levels in Cynomolgous monkey after administration of 0.1 mg/kganti-CSF-1R antibody. FIG. 4B: CSF-levels in Cynomolgous monkey afteradministration of 1 mg/kg anti-CSF-1R antibody. FIG. 4C: CSF-1 levels inCynomolgous monkey after administration of 10 mg/kg anti-CSF-1Rantibody. FIG. 4D: CSF-1 levels in Cynomolgous monkey afteradministration of 100 mg/kg anti-CSF-1R antibody.

FIG. 5A-5B illustrates data demonstrating human monocyte differentiationinto macrophages. FIG. 5A: Human Monocytes differentiated intomacrophages with coculture of GM-CSF or CSF-1 (100 ng/ml ligand). After6 days differentiation addition of R07155. Cell viability was measuredat day 7 of antibody treatment in a CTG Viability Assay (CellTiterGlo®Promega). Calculation of % cell viability: RLU signals from treatedcells divided by RLU signal from untreated control without antibody,(n=4). FIG. 5B: Human Monocytes differentiated into macrophages withGM-CSF (M1) or M-CSF (M2) for 7 days. Phenotype analyzed by indirectfluorescence analysis—staining with anti CD163-PE, anti CD80-PE or antiHLA-DR/DQ/DP-Zenon-Alexa647 labeled. The number in each histogramcorresponds to mean ratio fluorescence intensity (MRFI); calculatedratio between mean fluorescence intensity (MFI) of cells stained withthe selected antibody (empty histogram) and of corresponding isotypcontrol (negative control; gray filled histogram) (mean±SD; n>5).

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly it has been found that addition of TLR9 agonist toanti-CSF-1R antibody therapy resulted in a statistically significantmore than additive improvement of median time to progression compared toanti-CSF-1R antibody monotherapy or TLR9 agonist monotherapy (seeExample 13 and FIG. 1).

Many tumors are characterized by a prominent immune cell infiltrate,including macrophages. Initially, the immune cells were thought to bepart of a defense mechanism against the tumor, but recent data supportthe notion that several immune cell populations including macrophagesmay, in fact, promote tumor progression. Macrophages are characterizedby their plasticity. Depending on the cytokine microenvironment,macrophages can exhibit so-called M1 or M2-subtypes. M2 macrophages areengaged in the suppression of tumor immunity. They also play animportant role in tissue repair functions such as angiogenesis andtissue remodeling which are coopted by the tumor to support growth. Incontrast to tumor promoting M2 macrophages, M1 macrophages exhibitantitumor activity via the secretion of inflammatory cytokines and theirengagement in antigen presentation and phagocytosis (Mantovani, A. etal., Curr. Opin. Immunol. 2 (2010) 231-237).

By secreting various cytokines such as colony stimulating factor 1(CSF-1) and IL-10, tumor cells are able to recruit and shape macrophagesinto the M2-subtype, whereas cytokines such as granulocyte macrophagecolony stimulating factor (GM-CSF), IFN-gamma program macrophagestowards the M1 subtype. Using immunohistochemistry, it is possible todistinguish between a macrophage subpopulation co-expressing CD68 andCD163, which is likely to be enriched for M2 Macrophages, and a subsetshowing the CD68+/MHC II+, or CD68+/CD80+ immunophenotype, likely toinclude M1 macrophages. Cell shape, size, and spatial distribution ofCD68 and CD163 positive macrophages is consistent with publishedhypotheses on a tumor-promoting role of M2 macrophages, for example bytheir preferential location in tumor intersecting stroma, and vitaltumor areas. In contrast, CD68+/MHC class II+ macrophages areubiquitously found. Their hypothetical role in phagocytosis is reflectedby clusters of the CD68+/MHC class II+, but CD163-immunophenotype nearapoptotic cells and necrotic tumor areas.

The subtype and marker expression of different macrophage subpopulationsis linked with their functional state. M2 macrophages can supporttumorigenesis by:

-   -   a) enhancing angiogenesis via the secretion of angiogenic        factors such as VEGF or bFGF,    -   b) supporting metastasis formation via secretion of matrix        metalloproteinases (MMPs), growth factors and migratory factors        guiding the tumor cells to the blood stream and setting up the        metastatic niche (Wyckoff, J. et al., Cancer Res. 67 (2007)        2649-2656),    -   c) playing a role in building an immunosuppressive milieu by        secreting immunosuppressive cytokines such as IL-4, 11-13,        IL-1ra and IL-10, which in turn regulate T regulatory cell        function. Conversely CD4 positive T cells have been shown to        enhance the activity of tumor promoting macrophages in        preclinical models (Mantovani, A. et al., Eur. J. Cancer        40 (2004) 1660-1667; DeNardo, D. et al., Cancer Cell 16 (2009)        91-102).

Accordingly, in several types of cancer (e.g. breast, ovarian, Hodgkin'slymphoma) the prevalence of M2 subtype tumor associated macrophages(TAMs) has been associated with poor prognosis (Bingle, L. et al., J.Pathol. 3 (2002) 254-265; Orre, M., and Rogers, P. A., Gynecol. Oncol. 1(1999) 47-50; Steidl, C. et al., N. Engl. J. Med. 10 (2010) 875-885).Recent data show a correlation of CD163 positive macrophage infiltratein tumors and tumor grade (Kawamura, K. et al., Pathol. Int. 59 (2009)300-305). TAMs isolated from patient tumors had a tolerant phenotype andwere not cytotoxic to tumor cells (Mantovani, A. et al., Eur. J. Cancer40 (2004) 1660-1667). However, infiltration of TAMs in the presence ofcytotoxic T cells correlates with improved survival in non small celllung cancer and hence reflects a more prominent M1 macrophage infiltratein this tumor type (Kawai, O. et al., Cancer 6 (2008) 1387-1395).

Recently, a so-called immune signature comprising high numbers ofmacrophages and CD4 positive T cells, but low numbers of cytotoxic CD8positive T cells was shown to correlate with reduced overall survival(OS) in breast cancer patients and to represent an independentprognostic factor (DeNardo, D. et al., Cancer Discovery 1 (2011) 54-67).

Consistent with a role for CSF-1 in driving the pro-tumorigenic functionof M2 macrophages, high CSF-1 expression in rare sarcomas or locallyaggressive connective tissue tumors, such as pigmented villonodularsynovitis (PVNS) and tenosynovial giant cell tumor (TGCT) due in part toa translocation of the CSF-1 gene, leads to the accumulation ofmonocytes and macrophages expressing the receptor for CSF-1, thecolony-stimulating factor 1 receptor (CSF-1R) forming the majority ofthe tumor mass (West, R. B. et al., Proc. Natl. Acad. Sci. USA 3 (2006)690-695). These tumors were subsequently used to define a CSF-1dependent macrophage signature by gene expression profiling. In breastcancer and leiomyosarcoma patient tumors this CSF-1 response genesignature predicts poor prognosis (Espinosa, I. et al., Am. J. Pathol. 6(2009) 2347-2356; Beck, A. et al., Clin. Cancer Res. 3 (2009) 778-787).

CSF-1R belongs to the class III subfamily of receptor tyrosine kinasesand is encoded by the c-fms proto-oncogene. Binding of CSF-1 or IL-34induces receptor dimerization, followed by autophosphorylation andactivation of downstream signaling cascades. Activation of CSF-1Rregulates the survival, proliferation and differentiation of monocytesand macrophages (Xiong, Y. et al., J. Biol. Chem. 286 (2011) 952-960).

In addition to cells of the monocytic lineage and osteoclasts, whichderive from the same hematopoetic precursor as the macrophage,CSF-1R/c-fms has also been found to be expressed by several humanepithelial cancers such as ovarian and breast cancer and inleiomyosarcoma and TGCT/PVNS, albeit at lower expression levels comparedto macrophages. As with TGCT/PVNS, elevated levels of CSF-1, the ligandfor CSF-1R, in serum as well as ascites of ovarian cancer patients havebeen correlated with poor prognosis (Scholl, S. et al., Br. J. Cancer 62(1994) 342-346; Price, F. et al., Am. J. Obstet. Gynecol. 168 (1993)520-527). Furthermore, a constitutively active mutant form of CSF 1R isable to transform NIH3T3 cells, one of the properties of an oncogene(Chambers, S., Future Oncol 5 (2009) 1429-1440).

Preclinical models provide validation of CSF-1R as an oncology target.Blockade of CSF-1 as well as CSF-1R activity results in reducedrecruitment of TAMs. Chemotherapy resulted in elevated CSF-1 expressionin tumor cells leading to enhanced TAM recruitment. Blockade of CSF-1Rin combination with paclitaxel resulted in activation of CD8 positivecytotoxic T cells leading to reduced tumor growth and metastatic burdenin a spontaneous transgenic breast cancer model (DeNardo, D. et al.,Cancer Discovery 1 (2011) 54-67).

In one embodiment the invention comprises the combination therapy of anantibody binding to human CSF-1R, characterized in that the antibodybinds to human CSF-1R Extracellular Domain (SEQ ID NO: 64) incombination with a TLR9 agonist for use in the treatment of cancer.

In one embodiment the invention comprises the combination therapy of anantibody binding to human CSF-1R, characterized in that the antibodybinds to human CSF-1R Extracellular Domain (SEQ ID NO: 64) (comprisingdomains D1 to D5) and does not bind to domains D1 to D3 (SEQ ID NO: 66)of the extracellular domain of human CSF-1R in combination with a TLR9agonist for use in the treatment of cancer.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:7 and the light        chain variable domain is SEQ ID NO:8,    -   b) the heavy chain variable domain is SEQ ID NO:15 and the light        chain variable domain is SEQ ID NO:16;    -   c) the heavy chain variable domain is SEQ ID NO:75 and the light        chain variable domain is SEQ ID NO:76;    -   d) the heavy chain variable domain is SEQ ID NO:83 and the light        chain variable domain is SEQ ID NO:84;        or a humanized version thereof

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24, or    -   b) the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32, or    -   c) the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40, or    -   d) the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48, or    -   e) the heavy chain variable domain is SEQ ID NO:55 and the light        chain variable domain is SEQ ID NO:56.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24, or    -   b) the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32, or    -   c) the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40, or    -   d) the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:15 and the light        chain variable domain is SEQ ID NO:16, or a humanized version        thereof

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:75 and the light        chain variable domain is SEQ ID NO:76;    -   or a humanized version thereof

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:83 and the light        chain variable domain is SEQ ID NO:84;        or a humanized version thereof

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of        SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of        SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54; or    -   h) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region        of SEQ ID NO:71, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO:73, and        a CDR1 region of SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region        of SEQ ID NO: 79, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:80, a CDR2 region of SEQ ID NO: 81,        and a CDR1 region of SEQ ID NO: 82.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region        of SEQ ID NO:71, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO:73, and        a CDR1 region of SEQ ID NO:74, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region        of SEQ ID NO: 79, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:80, a CDR2 region of SEQ ID NO: 81,        and a CDR1 region of SEQ ID NO:

82.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of        SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of        SEQ ID NO: 27, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29, and        a CDR1 region of SEQ ID NO: 30.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of        SEQ ID NO: 35, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37, and        a CDR1 region of SEQ ID NO: 38.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of        SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that the antibody binds to humanCSF-1R fragment delD4 (SEQ ID NO: 65) and to human CSF-1R-ECD (SEQ IDNO: 64) with a ratio of 1:50 or lower, is further characterized in notbinding to human CSF-1R fragment D1-D3 (SEQ ID NO: 66).

The term “antibody” encompasses the various forms of antibodiesincluding but not being limited to whole antibodies, antibody fragments,human antibodies, humanized antibodies, chimeric antibodies, T cellepitope depleted antibodies, and further genetically engineeredantibodies as long as the characteristic properties according to theinvention are retained. “Antibody fragments” comprise a portion of afull length antibody, preferably the variable domain thereof, or atleast the antigen binding site thereof. Examples of antibody fragmentsinclude diabodies, single-chain antibody molecules, and multispecificantibodies formed from antibody fragments. scFv antibodies are, e.g.,described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-88). Inaddition, antibody fragments comprise single chain polypeptides havingthe characteristics of a V_(H) domain binding to CSF-1R, namely beingable to assemble together with a V_(L) domain, or of a V_(L) domainbinding to CSF-1R, namely being able to assemble together with a V_(H)domain to a functional antigen binding site and thereby providing theproperty.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from mouse and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a mouse variable region and a human constant region areespecially preferred. Such rat/human chimeric antibodies are the productof expressed immunoglobulin genes comprising DNA segments encoding ratimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of “chimeric antibodies”encompassed by the present invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies.” Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., Morrison, S. L., et al., Proc.Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 andU.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See e.g.Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Optionally the framework region canbe modified by further mutations. Also the CDRs can be modified by oneor more mutations to generate antibodies according to the invention e.g.by mutagenesis based upon molecular modeling as described by Riechmann,L., et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl.Acad. Sci. USA 86 (1989) 10029-10033, or others. Particularly preferredCDRs correspond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. A “humanized version of an antibodyaccording to the invention” (which is e.g. of mouse origin) refers to anantibody, which is based on the mouse antibody sequences in which theV_(H) and V_(L) are humanized by standard techniques (including CDRgrafting and optionally subsequent mutagenesis of certain amino acids inthe framework region and the CDRs). Preferably such humanized version ischimerized with a human constant region (see e.g. Sequences SEQ IDNO:57-61).

Other forms of “humanized antibodies” encompassed by the presentinvention are those in which the constant region has been additionallymodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding.

In the following examples the terms “Mab” or “muMab” refer to murinemonoclonal antibodies such as Mab 2F11 or Mab 2E10, whereas the term“hMab” refers to humanized monoclonal versions of such murine antibodiessuch as hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, etc.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G. J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, et al., andBoerner, et al., are also available for the preparation of humanmonoclonal antibodies (Cole, S. P. C., et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications”, nucleotide andamino acid sequence modifications which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-CSF-1Rantibody can be preferably replaced with another amino acid residue fromthe same side chain family.

Amino acid substitutions can be performed by mutagenesis based uponmolecular modeling as described by Riechmann, L., et al., Nature 332(1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033.

The human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor; Macrophagecolony-stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ IDNO: 22)) is known since 1986 (Coussens, L., et al., Nature 320 (1986)277-280). CSF-1R is a growth factor and encoded by the c-fmsproto-oncogene (reviewed e.g. in Roth, P. and Stanley, E. R., Curr. Top.Microbiol. Immunol. 181 (1992) 141-167).

CSF-1R is the receptor for the CSF-1R ligands CSF-1 (macrophage colonystimulating factor, also called M-CSF) (SEQ ID No.: 86) and IL-34 (SEQID No.: 87) and mediates the biological effects of these cytokines(Sherr, C. J., et al., Cell 41 (1985) 665-676; Lin, H., et al., Science320 (2008) 807-811). The cloning of the colony stimulating factor-1receptor (also called c-fms) was described for the first time inRoussel, M. F., et al., Nature 325 (1987) 549-552. In that publication,it was shown that CSF-1R had transforming potential dependent on changesin the C-terminal tail of the protein including the loss of theinhibitory tyrosine 969 phosphorylation which binds Cb1 and therebyregulates receptor down regulation (Lee, P. S., et al., Embo J. 18(1999) 3616-3628).

CSF-1R is a single chain, transmembrane receptor tyrosine kinase (RTK)and a member of the family of immunoglobulin (Ig) motif containing RTKscharacterized by 5 repeated Ig-like subdomains D1-D5 in theextracellular domain (ECD) of the receptor (Wang, Z., et al Molecularand Cellular Biology 13 (1993) 5348-5359). The human CSF-1RExtracellular Domain (CSF-1R-ECD) (SEQ ID NO: 64) comprises all fiveextracellular Ig-like subdomains D1-D5. The human CSF-1R fragment delD4(SEQ ID NO: 65) comprises the extracellular Ig-like subdomains D1-D3 andD5, but is missing the D4 subdomain. The human CSF-1R fragment D1-D3(SEQ ID NO: 66) comprises the respective subdomains D1-D3. The sequencesare listed without the signal peptide MGSGPGVLLL LLVATAWHGQ G (SEQ IDNO: 67). The human CSF-1R fragment D4-D3 (SEQ ID NO: 85) comprises therespective subdomains D4-D3.

Currently two CSF-1R ligands that bind to the extracellular domain ofCSF-1R are known. The first one is CSF-1 (colony stimulating factor 1,also called M-CSF, macrophage; human CSF-1, SEQ ID NO: 86) and is foundextracellularly as a disulfide-linked homodimer (Stanley, E. R. et al.,Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, E. R. etal., Stem Cells 12 Suppl. 1 (1995) 15-24). The second one is IL-34(human IL-34; SEQ ID NO: 87) (Hume, D. A., et al, Blood 119 (2012)1810-1820). Thus in one embodiment the term “CSF-1R ligand” refers tohuman CSF-1 (SEQ ID NO: 86) and/or human IL-34 (SEQ ID NO: 87).

For experiments often the active 149 amino acid (aa) fragment of humanCSF-1 (aa 33-181 of SEQ ID NO: 86) is used. This active 149 aa fragmentof human CSF-1 (aa 33-181 of SEQ ID NO: 86) is contained in all 3 majorforms of CSF-1 and is sufficient to mediate binding to CSF-1R (Hume, D.A., et al, Blood 119 (2012) 1810-1820).

The main biological effects of CSF-1R signaling are the differentiation,proliferation, migration, and survival of hematopoietic precursor cellsto the macrophage lineage (including osteoclast). Activation of CSF-1Ris mediated by its CSF-1R ligands, CSF-1 (M-CSF) and IL-34. Binding ofCSF-1 (M-CSF) to CSF-1R induces the formation of homodimers andactivation of the kinase by tyrosine phosphorylation (Li, W. et al, EMBOJournal. 10 (1991) 277-288; Stanley, E. R., et al., Mol. Reprod. Dev. 46(1997) 4-10).

The intracellular protein tyrosine kinase domain is interrupted by aunique insert domain that is also present in the other related RTK classIII family members that include the platelet derived growth factorreceptors (PDGFR), stem cell growth factor receptor (c-Kit) andfins-like cytokine receptor (FLT3). In spite of the structural homologyamong this family of growth factor receptors, they have distincttissue-specific functions.

CSF-1R is mainly expressed on cells of the monocytic lineage and in thefemale reproductive tract and placenta. In addition expression of CSF-1Rhas been reported in Langerhans cells in skin, a subset of smooth musclecells (Inaba, T., et al., J. Biol. Chem. 267 (1992) 5693-5699), B cells(Baker, A. H., et al., Oncogene 8 (1993) 371-378) and microglia (Sawada,M., et al., Brain Res. 509 (1990) 119-124). Cells with mutant humanCSF-1R ((SEQ ID NO: 23) are known to proliferate independently of ligandstimulation.

As used herein, “binding to human CSF-1R” or “specifically binding tohuman CSF-1R” or “specifically binds to human CSF-1R” or “which binds tohuman CSF-1R” or “anti-CSF-1R antibody” refers to an antibodyspecifically binding to the human CSF-1R antigen with a binding affinityof KD-value of 1.0×10⁻⁸ mol/l or lower at 35° C., in one embodiment of aKD-value of 1.0×10⁻⁹ mol/l or lower at 35° C. The binding affinity isdetermined with a standard binding assay at 35° C., such as surfaceplasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden) Amethod for determining the KD-value of the binding affinity is describedin Example 4. Thus an “antibody binding to human CSF-1R” as used hereinrefers to an antibody specifically binding to the human CSF-1R antigenwith a binding affinity of KD 1.0×10⁻⁸ mol/l or lower (preferably1.0×10⁻⁸ mol/l-1.0×10⁻¹² mol/l) at 35° C., preferably of a KD 1.0×10⁻⁹mol/l or lower at 35° C. (preferably 1.0×10⁻⁹ mol/l-1.0×10⁻¹² mol/l).

The “binding to human CSF-1R fragment delD4 (SEQ ID NO: 65) and to humanCSF-1R Extracellular Domain (SEQ ID NO: 64)” as used herein is measuredby a Surface Plasmon Resonance assay (Biacore assay) as described inExample 4. The human CSF-1R fragment delD4 (SEQ ID NO: 65) or humanCSF-1R Extracellular Domain (SEQ ID NO: 64), respectively, are capturedto the surface (each to a separate surface) and the test antibodies wereadded (each in a separate measurement) and the respective bindingsignals (Response Units (RU)) were determined. Reference signals (blanksurface) were subtracted. If signals of nonbinding test antibodies wereslightly below 0 the values were set as 0. Then the ratio of therespective binding signals (binding signal (RU) to human CSF-1R fragmentdelD4/binding signal (RU) to human CSF-1R Extracellular Domain(CSF-1R-ECD)) is determined. The antibodies according to the inventionhave a ratio of the binding signals (RU(delD4)/RU(CSF-1R-ECD) of 1:50 orlower, preferably of 1:100 or lower (the lower included end is 0 (e.g.if the RU is 0, then the ratio is 0:50 or 0:100)).

This means that such anti-CSF-1R antibodies according to the inventiondo not bind to the human CSF-1R fragment delD4 (like the anti-CCR5antibody m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18.08.2004 atDSMZ) and have binding signals for binding to the human CSF-1R fragmentdelD4 in the range of the anti-CCR5 antibody m<CCR5>Pz03.1C5, which arebelow 20 RU (Response Units), preferably below 10 RU in a SurfacePlasmon Resonance (BIAcore) assay as shown in Example 4.

The term “binding to human CSF-1R fragment D1-D3” refers to a bindingaffinity determination by a Surface Plasmon Resonance assay (Biacoreassay). The test antibody is captured to the surface and the humanCSF-1R fragment D1-D3 (SEQ ID NO: 66) was added and the respectivebinding affinities were determined. The terms “not binding to humanCSF-1R fragment D1-D3” or “which do not bind to human CSF-1R fragmentD1-D3” denotes that in such an assay the detected signal was in the areaof no more than 1.2 fold of background signal and therefore nosignificant binding could be detected and no binding affinity could bedetermined (see Example 10).

The term “ligand dependent” as used herein refers to aligand-independent signaling through the extracellular ECD (and does notinclude the ligand independent signaling mediated by activating pointmutations in the intracellular kinase domain).

In one embodiment CSF-1R ligand in this context refers a CSF-1R ligandselected from human CSF-1 (SEQ ID No: 86) and human IL-34 (SEQ ID No:87); in one embodiment the CSF-1R ligand is human CSF-1 (SEQ ID No: 86);in one embodiment the CSF-1R ligand is human IL-34 (SEQ ID No: 87)).

The invention comprises an antibody binding to human CSF-1R, antibodybinding to human CSF-1R, for use in the treatment of a patient having aCSF-1R expressing tumor or having a tumor with CSF-1R expressingmacrophage infiltrate, wherein the tumor is characterized by an increaseof CSF-1R ligand (in one embodiment the CSF-1R ligand is selected fromhuman CSF-1 (SEQ ID No: 86) and human IL-34 (SEQ ID No: 87); in oneembodiment the CSF-1R ligand is human CSF-1 (SEQ ID No: 86); in oneembodiment the CSF-1R ligand is human IL-34 (SEQ ID No: 87)) (detectablein serum, urine or tumor biopsies), wherein the anti-CSF-1R antibody isadministered in combination with TLR9 agonist. (In one embodiment theCSF-1R antibody is characterized in binding to the (dimerization)domains D4 to D5 (SEQ ID No: 85) of the extracellular domain of humanCSF-1R.

The term “increase of CSF-1R ligand” refers to the overexpression ofhuman CSF-1R ligand (in one embodiment the CSF-1R ligand is selectedfrom human CSF-1 (SEQ ID No: 86) and human IL-34 (SEQ ID No: 87); in oneembodiment the CSF-1R ligand is human CSF-1 (SEQ ID No: 86); in oneembodiment the CSF-1R ligand is human IL-34 (SEQ ID No: 87)) (comparedto normal tissue) before treatment or overexpression of human CSF-1Rligand induced by treatment with anti-CSF-1R antibody (and compared tothe expression levels before treatment). In certain embodiments, theterm “increase” or “above” refers to a level above the reference levelor to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, 100% or greater, in CSF-1R ligand level detected bythe methods described herein, as compared to the CSF-1R ligand levelfrom a reference sample. In certain embodiments, the term increaserefers to the increase in CSF-1R ligand level wherein, the increase isat least about 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-,20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold higher ascompared to the CSF-1R ligand level e.g. predetermined from a referencesample. In one preferred embodiment the term increased level relates toa value at or above a reference level.

In one embodiment of the invention the anti-CSF-1R antibody ischaracterized in that the antibody binds to human CSF-1R ExtracellularDomain (SEQ ID NO: 64) (comprising domains D1 to D5) and binds todomains D1 to D3 (SEQ ID NO: 66) of the extracellular domain of humanCSF-1R.

In one embodiment of the invention the anti-CSF-1R antibody ischaracterized in that the antibody binds to human CSF-1R ExtracellularDomain (SEQ ID NO: 64) (comprising domains D1 to D5) and does not bindto domains D1 to D3 (SEQ ID NO: 66) of the extracellular domain of humanCSF-1R.

The term “Toll-like receptor 9” (TLR9, CD289; SEQ ID NO: 88) refers to aprotein of the Toll-like receptor (TLR) family which plays a fundamentalrole in pathogen recognition and activation of innate immunity. TLRs arehighly conserved from Drosophila to humans and share structural andfunctional similarities. They recognize pathogen-associated molecularpatterns (PAMPs) that are expressed on infectious agents, and mediatethe production of cytokines necessary for the development of effectiveimmunity. The various TLRs exhibit different patterns of expression.This gene is preferentially expressed in immune cell rich tissues, suchas spleen, lymph node, bone marrow and peripheral blood leukocytes.Studies in mice and human indicate that this receptor mediates cellularresponse to unmethylated CpG dinucleotides in bacterial DNA to mount aninnate immune response.

TLR9 is mainly found in the endosomal compartment of B cells, monocytes,macrophages and plasmacytoid Dendritic Cells DCs (Galluzzi et al.,Oncolmmunology, 1:5, (2012) 699-716). The main ligand of TLR9 isbacterial/viral DNA, differing from its mammalian counterpart for theelevated frequency of unmethylated CpG oligodeoxynucleotides. Indeed,whereas mammalian DNA has no immunostimulatory activity, theadministration of bacterial/viral DNA induces a potent Th1 immuneresponse in vivo, which is entirely abrogated in TLR9^(−/−) mice. CpGoligodeoxynucleotides (or CpG ODN) are short single-stranded syntheticDNA molecules that contain a cytidine triphosphate deoxynucleotide (“C)”followed by a guanidine triphosphate deoxynucleotide (“G”). The “p”refers to the phosphodiester link between consecutive nucleotides,although some ODN have a modified phosphorothioate (PS) backboneinstead. When these CpG motifs are unmethlyated, they act asimmunostimulants (Weiner, G J; et al, PNAS 94 (1997) 10833-7). Thus“Toll-like receptor 9 agonists” (TLR9 agonist) are characterized inbinding to Toll-like receptor 9 and in stimulating TLR9 immune response.E.g. in one embodiment a Toll-like receptor 9 agonist (TLR9 agonist) ischaracterized by binding to Toll-like receptor 9 on human plasmacytoiddendritic cells (pDCs) and by induction of IFN-alpha, IL-6, and/or IL-12(elevating the levels of IFN-alpha, IL-6, and/or IL-12) in theseplasmacytoid dendritic cells (pDCs).

CpG motifs are considered pathogen-associated molecular patterns (PAMPs)due to their abundance in microbial genomes but their rarity invertebrate genomes (Bauer, S; Current Topics in Microbiology andImmunology 270 (2002) 145-54). The CpG PAMP is recognized by the patternrecognition receptor (PRR) Toll-Like Receptor 9 (TLR9), which isconstitutively expressed only in B cells and plasmacytoid dendriticcells (pDCs) in humans and other higher primates (Rothenfusser, S; etal, Human immunology 63 (2002) 1111-9)

Synthetic CpG ODN differ from microbial DNA in that they have apartially or completely phosphorothioated (PS) backbone instead of thetypical phosphodiester backbone and a poly G tail at the 3′ end, 5′ end,or both. PS modification protects the ODN from being degraded bynucleases such as DNase in the body and poly G tail enhances cellularuptake (Dalpke, A H et al, Immunology 106 (2002) 102-12). The poly Gtails form intermolecular tetrads that result in high molecular weightaggregates. These aggregates are responsible for the increased activitythe poly G sequence impart; not the sequence itself

These synthetic oligodeoxynucleotides containing unmethylated CpG motifs(CpG ODNs), such as ODN 1826, have been extensively studied as adjuvants(Steinhagen F. et al., 2011; Vaccine 29(17):3341-55). These CpG motifsare present at a 20-fold greater frequency in bacterial DNA compared tomammalian DNA (Hemmi H. et al., 2000. Nature 408: 740-5). CpG ODNsagonize TLR9, which is expressed on human B cells and plasmacytoiddendritic cells (pDCs), thereby inducing Th1-dominated immune responses(Coffman et al., 2010. Immunity 33(4):492-503). Pre-clinical studies,conducted in rodents and non-human primates, and human clinical trialshave demonstrated that CpG ODNs can significantly improvevaccine-specific antibody responses (Steinhagen F. et al., 2011; Vaccine29(17):3341-55).

Numerous sequences have been shown to stimulate TLR9 with variations inthe number and location of CpG dimers, as well as the precise basesequences flanking the CpG dimers. This led to the creation of classesor categories of CpG ODN, which are all TLR9 agonist based on theirsequence, secondary structures, and effect on human peripheral bloodmononuclear cells (PBMCs). The three main classes of CpG ODNs are classA, B and C, which differ in their immune-stimulatory activities (Krug A.et al., 2001, Eur J Immunol, 31(7): 2154-63). Furthermore, CpG ODNsactivate TLR9 in a species-specific manner (Bauer, S. et al., 2001,PNAS, 98(16):9237-42). One of the first Class A ODN, ODN 2216, wasdescribed in 2001 by Krug et al (see above) This class of ODN wasdistinctly different from the previously described Class B ODN (i.e.,ODN 2006) in that it stimulated the production of large amounts of TypeI interferons, the most important one being IFNα, and induced thematuration of pDCs.

Class A ODN are also strong activators of NK cells through indirectcytokine signaling. Class A ODN typically contain 7 to 10 PS-modifiedbases at one or both ends that resist degradation by nucleases andincrease the longevity of the ODN. The above rules strictly define theclass, but variability of the sequence within these “rules” is possible.It should also be noted that changes to the sequence will affect themagnitude of the response. For example, the internal palindrome sequencecan be 4 to 8 base pairs in length and vary in the order of bases,however the pattern, 5′-Pu Pu CG Pu Py CG Py Py-3′, was found to be themost active when compared to several other sequences. The poly G tailfound at either end of the DNA strand can vary in length and evennumber, but its presence is critical to the activity of the molecule.

Class B ODN (i.e. ODN 2007) are strong stimulators of human B cell andmonocyte maturation. They also stimulate the maturation of pDC but to alesser extent than Class A ODN and very small amounts of IFNα. Thestrongest ODN in this class have three 6 mer sequences. Class B ODNshave been studied extensively as therapeutic agents because of theirability to induce a strong humoral immune response, making them ideal asa vaccine adjuvant.

ODN 1826 is a type B CpG ODN specific for mouse TLR9. Type B CpG ODNscontain a full phosphorothioate backbone with one or more CpGdinucleotides and can strongly activate B cells (Krug A. et al., 2001,Eur J Immunol, 31(7): 2154-63). ODN 1826, a mouse-reactive surrogateTLR9-agonist has been tested as an adjuvant in numerous animal models(Bauer, S. et al., 2001, PNAS, 98(16):9237-42). Research in micedemonstrated that ODN 1826 administration can induce the activation ofantigen presenting cells and type I IFN anti-viral activity 8-9,indicative of a Th1 immune response (Longhi Mp. et al., 2009, J Exp Med206: 1589-602).

Moreover, the administration of type B CpG oligonucleotides (alone orcombined with chemotherapeutics or peptide vaccines) to tumor-bearingrodents reportedly exerts potent anticancer effects. Initial Phase I/IIclinical trials to test the safety and efficacy of CpG-7909 foroncological indications were launched in April 2000. Approximately inthe same period, CpG-7909 begun to be extensively investigated as anadjuvant for cancer-unrelated indications (mainly antiviral vaccines),showing no severe side effects and encouraging efficacy.

During the last decade, the safety and anticancer potential of CpG-7909(as a standalone agent or in combination with chemotherapy and/orvaccination approaches) have been investigated in a large number ofPhase I/II clinical trials, including studies with leukemia, lymphoma,basal cell carcinoma, lmelanoma, esophageal squamous cell carcinoma,NSCLC, renal cell carcinoma, and prostate cancer patients. Several TLR9agonist are known and currently developed in clinical testing Agatolimod(tricosasodium salt of a synthetic 24-mer oligonucleotide containing 3CpG motifs; Pfizer) GNKG168 (CpG ODN; SBI Biotech), IMO-2055 (syntheticoligonucleotide containing unmethylated CpG dinucleotides; IderaPharmaceuticals), MGN-1703 (Mologen). Typically these TLR9 agonist areused in the treatment of different cancers:

Bacterial and synthetic DNA containing unmethylated CpG motifs act asagonists of TLR9 and induce Th1-type immune response profiles. Theimmune-stimulatory effects of TLR9 agonists are multifactorial anddepend on the nucleotide sequence, the nature of the backbone and thepresence of specific structural motifs. Based on the cytokine profilesinduced, three distinct types of TLR9 agonists, class A, B and C, havebeen described. Each class of TLR9 agonist is composed of a differentnucleotide sequence that allows formation of structures (or nostructures) that generate different immune profiles.

The structure-activity relationships of oligonucleotides that act asagonists of TLR9 was systematically studied (Kandimalla, E. R. andAgrawal, S. (2005) in Toll and Toll Receptors: An ImmunologicPerspective (Rich, T., ed.), pp. 181-212, Kluwer Academic/PlenumPublishers, New York). The presence of a CpG motif in oligonucleotidesis required for TLR9 stimulation. Oligonucleotides with phosphodiesterand phosphorothioate backbone stimulate TLR9-mediated immune responses.

Phosphorothioate backbone oligonucleotides are commonly used becausethey are less susceptible to degradation by ubiquitous nucleases thanare phosphodiester oligonucleotides. Introduction of a sulfur atom onthe internucleotide phosphodiester bond results in the formation of Rpand Sp diastereoisomers; the Rp diastereomer of phosphorothioate linkagestimulates a stronger TLR9-mediated immune response than does the Spdiastereomer. The negative charges on phosphates between and adjacent tocytosine (C) and guanine (G) are also required for TLR9-mediatedactivity. Neutralization of charges by incorporation ofmethylphosphonate linkages at these positions results in the loss ofimmune-stimulatory activity. Moreover, TLR9 activation is also dependenton the sequences flanking the CpG dinucleotide, the nature of thenucleotide backbone and the secondary structures.

Flanking Sequences Play a Significant Role in TLR9 Stimulation

Chemical modifications introduced at the 2′-position of the sugar ringof a C or G nucleotide in the CpG motif result in the loss ofimmune-stimulatory activity of TLR9 agonists. In addition, studies ofTLR9 agonists containing chemical modifications such asmethylphosphonate linkages, 2′-alkyl or 3′-deoxy or -alkylribonucleosides, non-nucleotide linkers or abasic nucleotides in theflanking sequences indicate that substitutions incorporated at thefourth to sixth nucleotide positions 5′ to the CpG dinucleotidesignificantly enhance immune-stimulatory activity. In general,modifications incorporated in the 3′-flanking sequence distal to the CpGdinucleotide have effects dependent on the nature of the modification.

TLR9 Requires a Free 5′-End of Agonist for Stimulation

Two CpG oligonucleotides linked through their 5′-ends do not activateimmune cells despite the availability of two CpG motifs. When the sameoligonucleotides are linked through their 3′-ends, they produce higherand distinct cytokine profiles than the parent CpG oligonucleotide witha single 5′-end. These are the first studies demonstrating therequirement of an accessible or free 5′-end for TLR9 activation and thatthe receptor reads the sequence from the 5′-end. The transcriptionfactor NF-κB is rapidly activated by TLR9 agonists that contain two5′-ends, but these compounds have the same activity as conventional TLR9agonists on the MAPK (mitogen-activated protein kinase) pathway in J774cells.

These studies suggest that agonists containing two 5′-ends facilitatedimerization of the receptor, leading to rapid activation of immuneresponses. Moreover, TLR9 activation can be modulated throughappropriate presentation of the free 5′-ends and syntheticimmune-stimulatory motifs, leading to changes in the downstream cytokineinduction profiles. Consistent with these results, recent studies haveshown that TLR9 exists in dimer form and binds to single-strandedoligonucleotides. However, only oligonucleotides containing the CpGmotif cause conformational changes in the receptor, leading to theactivation of immune signalling pathways.

The attachment of oligonucleotides through their 3′-ends not onlyprovides two 5′-ends for optimal activation of TLR9, but also increasesthe stability against 3′-exonucleases. Oligonucleotides with aphosphodiester backbone and as short as 5 and 6 nt linked through their3′-ends act as potent TLR9 agonists and produce immune responses.Moreover, oral administration of the novel structure containing TLR9agonists induces potent mucosal immune responses, acts as an adjuvantwith antigens, and prevents and reverses peanut allergy in mouse modelsbecause of their greater stability in the gastrointestinal tract.

Functional Groups of Cytosine and Guanine Required for TLR9 Stimulation

As described above, certain chemical modifications introduced within theCpG dinucleotide that alter structure and conformation lead to the lossof immune-stimulatory activity of agonists. One such modification is areplacement of the methyl group at the 5-position of cytosine in the CpGmotif of TLR9 agonists. Vertebrates use this feature to distinguishself-DNA from that of bacterial DNA, which contains more unmethylatedCpG motifs.

The effects of various pyrimidine analogues (Y), such as 5-OH-dC, dU,dP, 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,N3-Me-dC and N4-Et-dC, in place of cytosine (Kandimalla, E. R., et al(2001) Bioorg. Med. Chem. 9, 807-813; Kandimalla, E. R., et al, S.(2003) PNAS. 100, 14303-14308; or Putta, M. R., et al, S. (2006) NucleicAcids Res. 34, 3231-3238). To understand the role of differentfunctional groups of guanine in the recognition of TLR9, several purinenucleobases (R) such as 7-deaza-dG, N1-Me-dG, 2-amino-D-purine,nebularine, 2-amino-dA, 7-deaza-D-xanthine, K-base and dI were examinedin place of guanine in the CpG (Kandimalla, E. R., et al (2001) Bioorg.Med. Chem. 9, 807-813; Kandimalla, E. R., et al. (2003) PNAS. 100,14303-14308; Putta, M. R., et al, (2006) Nucleic Acids Res. 34,3231-3238; Kandimalla, E. R., et al (2003) Nucleic Acids Res. 31,2393-2400; or Kandimalla, E. R., et al. (2005) PNAS. 102, 6925-6930).These studies led to the development of alternative synthetic nucleotidemotifs (YpG, CpR) for immune modulation and have demonstrated acceptanceby TLR9 of certain heterocyclic base variants.

Novel synthetic agonists of TLR9 (S. Agrawal and E. R. Kandimalla,Biochemical Society Transactions (2007) 35, (1461-1467)): Thecombinations of novel structures and synthetic immune-stimulatory motifsdescribed above provided us with tools to generate combinatoriallibraries of novel synthetic agonists of TLR9. Systematic studies ofseveral TLR9 agonists that have two 5′-ends and contain synthetic CpRdinucleotides in different nucleotide compositions in mouse, human andmonkey systems suggest that nucleotide sequence and secondary structuresplay a role in modulating the immune response. Based on these studies,we have broadly identified two different groups of synthetic agonists ofTLR9.

In one embodiment the TLR9 agonist is characterized by induction ofIFN-alpha, IL-6, and/or IL-12 (elevating the levels of IFN-alpha, IL-6,and/or IL-12) in plasmacytoid dendritic cells (pDCs). In one embodimentthe TLR9 agonist is characterized by elevating the level of IFN-alpha inhuman plasmacytoid dendritic cells (pDCs) (as measured by sandwich ELISAas described below or e.g in in WO2010/088395)

Assay for Measuring IFN-Alpha Induction (Elevating the Levels ofIFN-Alpha, IL-6, and/or IL-12) by TLR9 Agonist of the Invention in HumanpDCs:

Human PBMC isolation: Peripheral blood mononuclear cells (PBMCs) fromfreshly drawn healthy volunteer blood (CBR Laboratories, Boston, Mass.)are isolated by Ficoll density gradient centrifugation method(Histopaque-1077, Sigma).

Human pDC isolation: Human plasmacytoid dendritic cells (pDCs) areisolated from freshly obtained healthy human volunteer's blood PBMCs bypositive selection using the BDC A4 cell isolation kits (MiltenyiBiotec) according to the manufacturer's instructions.

Human pDCs are plated in 96-well dishes using 1×10⁶ cells/ml. Individualimmune modulatory compounds from Table I were dissolved in DPBS (pH 7.4;Mediatech) are added to the cell cultures at doses of 0, 0.1, 0.3, 1.0,3.0, or 10.0.micro.g/ml. The cells were then incubated at 37 (0)C for 24hoursand the supernatants were collected for luminex multiplex or ELISAassays.

In the levels of IFN-alpha, IL-6, and/or IL-12 are measured by sandwichELISA. The required reagents, including cytokine antibodies andstandards, can be purchased from PharMingen.

IFN-alpha has been known as an antiviral cytokine for many years. Itstimulates Th1 cell development, therefore promoting the effects ofCG-containing DNA molecules. IFN-alpha also exhibits antitumour activityin mouse and human malignancies and is capable of decreasing thetumourigenicity of transplanted tumour cells, partially by activatingcytotoxic T cells and thereby increasing the likelihood of tumour-cellcytolysis. NK cell and macrophage activity, both also important forantitumour cytotoxicity, are also increased by IFN-alpha (Brassard etal., J. Leukoc. Biol. 2002 71: 565-81). Therefore, increasing the amountof IFN-alpha upon stimulation with the DNA constructs of the presentdisclosure is expected to be beneficial for the treatment of cancer.

In one embodiment of the invention the TLR9 agonist of the invention isan oligodeoxynucleotide containing a) cytosine-phosphate-guanosine (CpG)motifs (CpG ODNs) b) pyrimidine-phosphate-guanosine (YpG) motifs (YpGODNs) or c) cytosine-phosphate-purine (CpR) motifs (CpR ODNs).

In one embodiment of the invention the TLR9 agonist of the invention isan oligodeoxynucleotide containing a) cytosine-phosphate-guanosine (CpG)motifs (CpG ODNs) b) pyrimidine-phosphate-guanosine (YpG) motifs (YpGODNs) or c) Purine-phosphate-guanosine (RpG) motifs (RpG ODNs) whereinthe TLR9 agonist stimulates TLR9 (in one embodiment the TLR9 agonistinduces the maturation of plasmacytoid dendritic cells (pDCs); in oneembodiment the TLR9 agonist is charcterized by human B cell maturation;in one embodiment)

In one embodiment of the invention the TLR9 agonist of the invention isan oligodeoxynucleotide containing cytosine-phosphate-guanosine (CpG)motifs (CpG ODNs).

In one embodiment of the invention the TLR9 agonist of the invention isa Class A CpG ODN.

In one embodiment the TLR9 agonist of the invention is anoligodeoxynucleotide comprising

a) a poly G sequence at the 5′ end, or the 3′ end, or at both ends

b) an internal palindrome sequence;

c) GC dinucleotides contained within the internal palindrome, and

d) a partially PS-modified backbone

Class A CpG ODN typically contain 7 to 10 PS-modified bases at one orboth ends that resist degradation by nucleases and increase thelongevity of the ODN. The above rules strictly define the class, butvariability of the sequence within these rules is possible. The internalpalindrome sequence can be 4 to 8 base pairs in length and vary in theorder of bases, however the pattern, 5′-Pu Pu CG Pu Py CG Py Py-3′, wasfound to be the most active when compared to several other sequences.The poly G tail found at either end of the DNA strand can vary in lengthand number.

In one embodiment the Class A CpG ODN (Xueqing Liang, et al, Blood. 2010June 17; 115(24): 5041-5052) is selected from the group consisting ofCpG ODN 2216 (5′-ggGGGACGATCGTCgggggG-3′) (SEQ ID NO: 89) CpG ODN PB4(5′-tcgGACGATCGTCgggggG-3′) (SEQ ID NO: 90); or CpG ODN 1002(5′-ggGGTCGTTCGTCGTTgggggG-3′) (SEQ ID NO: 91).

In one embodiment of the invention the TLR9 agonist of the invention isa Class B CpG ODN.

In one embodiment the TLR9 agonist of the invention is aoligodeoxynucleotides comprising

-   -   a) one or more 6 mer unmethylated cytosine-phosphate-guanosine        (CpG) motifs 5′-Pu Py C G Py Pu-3′ (one or more 6 mer        5′-RYCGYR-3′ 6-mers (R=A or G; Y=T or C))    -   b) a fully phosphorothioated (PS-modified) backbone; and    -   c) 18 to 28 nucleotides in length

In one embodiment the Class B CPG ODN is selected from the groupconsisting of CpG-28, CpG-685 (GNKG168; CpG ODN; SBI Biotech), CpG-684and CpG-7909 (CPG-ODN 2006, PF-3512676, Agatolimod).

CpG-7909 (CpG 2006, PF-3512676, Agatolimod) is a Synthetic, 24-merphosphothioate oligodeoxynucleotide(d(P-Thio)(T-C-G-T-C-G-T-T-T-T-G-T-C-G-T-T-T-T-G-T-C-G-T-T)DNA)(5′-tcgtcgttttgtcgttttgtcgtt-3′) (SEQ ID NO: 92) containing multiplecytosine-phosphate-guanosine (CpG) motifs or one of its derivatives liketricosasodium salt. The preparation is described e.g. in WO 9818810 orU.S. Pat. No. 7,223,741)

CpG-685 (GNKG168; CpG ODN; SBI Biotech) is synthetic, 21-mer,unmethylated CpG motif-based oligodeoxynucleotide (ODN) (685,5′-tcgtcgacgtcgttcgttctc-3′) (SEQ ID NO: 93), with immunostimulatoryactivity. CpG685 (GNKG168), a 21-mer fully phosphorothioatedoligonucleotides designed to directly target Toll-like receptor 9 thatmediates cellular responses in B cells, showed antitumor effects in SCIDmouse and is under clinical development for the treatment of humanchronic lymphocytic leukemia (B-CLL) by SBI Biotech Co. Herein, asensitive and specific assay was developed in plasma and cell lysate tosupport its preclinical pharmacology studies. CpG oligodeoxynucleotideGNKG168 binds to and activates Toll-like receptor 9 (TLR9) and is takenup into cells by endocytosis; once internalized, it may activatenumerous signaling transduction pathways resulting in the release ofmultiple cytokines, such as immunoglobulins (Igs), interferons (IFNs),interleukins (ILs) and tumor necrosis factor (TNF).

CpG-684 is synthetic, 23-mer, unmethylated CpG motif-basedoligodeoxynucleotide (ODN) 684, 5′-tcgacgttcgtcgttcgtcgttc-3′ (SEQ IDNO: 94);

CpG-28 synthetic unmethylated CpG motif-based oligodeoxynucleotide(ODN), containing multiple repeats of unmethylated CpG motifs (CpG ODN)with immunostimulatory activity (5′-TAAACGTTATAACGTTATGACGTCAT-3′) (SEQID NO: 95) with a wholly phosphorothioate backbone (Carpentier A F, etal Front Biosci. 2003; 8:e115-e127; Meng Y, et al, Int J Cancer. 2005;116:992-997; or Carpentier A, et al. Neuro-Oncology 2006; 8:60-66). Uponentering the cell via endocytosis, CpG-28 activates numerous signalingtransduction pathways resulting in the release of multiple cytokines.CpG-28 has immunomodulatory properties with direct activation ofB-lymphocytes, dendritic and NK cells resulting in the stimulation ofinnate immunity and antibody-dependant cell cytotoxicity (ADCC).Additionally, this agent indirectly modulates T-cell responses thoughthe release of cytokines (IL-12 and IFN gamma) to induce a preferentialshift to the Th1 (helper) phenotype resulting in enhanced CD8+ cellularcytotoxicity.

In one embodiment of the invention the TLR9 agonist of the invention isa oligodeoxynucleotides containing pyrimidine-phosphate-guanosine (YpG)motifs (YpG ODNs).

In one embodiment of the invention the TLR9 agonist of the invention isa oligodeoxynucleotides containing cytosine-phosphate-purine (CpR)motifs (CpR ODNs).

In one embodiment of the invention the TLR9 agonist of the invention isIMO-2055 (Idera) (ODN consisting of 3′-3′-linked structure and syntheticCpR(R=2′-deoxy-7-deazaguanosine) motif)

In one embodiment of the invention the TLR9 agonist of the invention isa oligodeoxynucleotides containing a) cytosine-phosphate-guanosine (CpG)motifs (CpG ODNs) b) pyrimidine-phosphate-guanosine (YpG) motifs (YpGODNs) or c) cytosine-phosphate-purine (CpR) motifs (CpR ODNs).

In one embodiment of the invention the TLR9 agonist of the invention isa oligodeoxynucleotides based CpG motif-containing circular ODN (e.gMGN-1703 from Mologen as described in WO2012/085291) based on the dSLIM®technology (this technology is described in WO2001/07055).

In one embodiment of the invention the TLR9 agonist is selected from thegroup consisting of CpG ODN 2216 CpG ODN 1002 CpG-28, CpG-685, CpG-684,CpG-7909, IMO-2055 or MGN-1703. In one embodiment of the invention theTLR9 agonist is selected from the group consisting of CpG-685, CpG-7909,IMO-2055 or MGN-1703. In one embodiment the TLR9 agonist is selectedfrom the group consisting of CpG-7909, IMO-2055 or MGN-1703.

In one embodiment of the invention the CSF-1R antibody is selected fromantibodies described in WO 2009/026303, WO 2009/112245,WO2011/123381(A1) or WO2011/070024; and the TLR9 agonist is selectedfrom the group consisting of CpG-685, CpG-7909, IMO-2055 or MGN-1703.

In one embodiment of the invention the CSF-1R antibody is selected fromantibodies binding to human CSF-1R, characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of        SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54;    -   and the TLR9 agonist is selected from the group consisting of        CpG-685, CpG-7909, IMO-2055 or MGN-1703

In one embodiment of the invention the CSF-1R antibody is selected fromantibodies binding to human CSF-1R, is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40.    -   and the TLR9 agonist is selected from the group consisting of        CpG-685, CpG-7909, IMO-2055 or MGN-1703

In general, many suitable TLR9 agonists are known in the art. These TLR9agonists are contemplated to be used for the present combination therapyof the invention. TLR9 specifically recognises CpG DNA that isunmethylated, and initiates a signalling cascade leading to theproduction of proinflammatory cytokines. Methylation of the cytosinewithin the CpG motif strongly reduces the affinity of TLR9. Doublestranded (ds) CpG DNA is a weaker stimulator of TLR9 compared to itssingle stranded (ss) counterpart.

Naturally occurring agonists of TLR9 are described in Smith & Wickstrom(1998) J. Natl. Cancer Inst. 90:1146-1154), and their role in cancer isdescribed in Damiano et al. (2007) Proc. Nat. Acad. Sci. USA 104:12468-12473.

CPG 7909 is an immunostimulatory TLR9 agonist oligodeoxynucleotide thatwas found to be well tolerated in a phase 1/1 I clinical study (Cooperet al, (2004) J. Clin. Immunol., 24(6): 693-701). The CpG enriched,synthetic oligodeoxynucleotide TLR9 agonist PF-3512676 was found to haveantilymphoma activity in a phase 1/1 I clinical study (Brody et al(2010) J. Clin. Oncol., 28(28): 4324-32).

Certain TLR9 agonists are comprised of 3-3′ linked DNA structurescontaining a core CpR dinucleotide, wherein the R is a modifiedguanosine (U.S. Pat. No. 7,276,489). In addition, specific chemicalmodifications have allowed the preparation of specific oligonucleotideanalogues that generate distinct modulations of the immune response. Inparticular, structure activity relationship studies have allowedidentification of synthetic motifs and novel DNA-based compounds thatgenerate specific modulations of the immune response and thesemodulations are distinct from those generated by unmethylated CpGdinucleotides (Kandimalla et al. (2005) Proc. Natl. Acad. Sci. USA 102:6925-6930; Kandimalla of al. (2003) Proc. Nat. Acad. Sci. USA 100:14303-14308; Cong et al. (2003) Biochem Biophys Res. Commun. 310:1133-1139; Kandimalla of al. (2003) Biochem. Biophys. Res. Commun 306:948-953; Kandimalla et al. (2003) Nucleic Acids Res. 31: 2393-2400; Yu,D. et al. (2003) Bioorg. Med. Chem. 1 1:459-464; Bhagat, L. et al.(2003) Biochem. Biophys. Res. Commun 300:853-861; Yu, D. et al. (2002)Nucleic Acids Res. 30:4460-4469; Yu, D. et al. (2002) J. Med. Chem.45:4540-4548. Yu, D. et al. (2002) Biochem. Biophys. Res. Commun297:83-90; Kandimalla. E. et al. (2002) Bioconjug. Chem. 13:966-974; Yu,D. et al. (2002) Nucleic Acids Res. 30:1613-1619; Yu, D. et al. (2001)Bioorg. Med. Chem. 9: 2803-2808; Yu ef al. (2001) Bioorg. Med. Chem.Lett. 1: 2263-2267; Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813; Yu ef al. (2000) Bioorg. Med. Chem. Lett. 10: 2585-2588; andPutta et al. (2006) Nucleic Acids Res. 34: 3231-3238).

US 2009/0053206 describes a number of TLR9 agonists, in particularcompounds 1-169 listed in Table 1; US 2008/0292648 describes a number ofTLR9 agonists, in particular compounds 1-92 listed in Table 1; and US2007/0105800 describes oligonucleotidebased compounds that are TLR9agonists (Idera Pharmaceuticals). Suitable TLR9 agonists may alsoinclude the selective TLR9 agonists IMO-2055, IMO-2125 and IMO-2134 thatare undergoing phase 1/phase 2 clinical trials (Idera Pharmaceuticals).US 2010/0016250 describes a number of TLR9 agonists, in particularcompounds of Formula I (Kyowa Hakko Kirin Co). As mentioned above, US2009/0041809 describes compositions that are TLR9 agonists or both TLR3and TLR9 agonists (Nventa Pharmaceuticals).

Different TLR9 agonists to be used for the present combination therapyof the invention are described in detail in WO2007/7047396,WO2007/7047396, WO2010088395, WO03035695, WO2012085291, WO 1998/018810,WO 2005/042018, WO2008073959, WO2009018431, WO2007084237.

The term “epitope” denotes a protein determinant of human CSF-1R capableof specifically binding to an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually epitopes have specific three dimensionalstructural characteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Preferably an antibody according to the inventionbinds specifically to native and to denatured CSF-1R.

The “variable domain” (variable domain of a light chain (V_(L)),variable domain of a heavy chain (V_(H))) as used herein denotes each ofthe pair of light and heavy chain domains which are involved directly inbinding the antibody to the antigen. The variable light and heavy chaindomains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementary determining regions,CDRs). The framework regions adopt a β-sheet conformation and the CDRsmay form loops connecting the β-sheet structure. The CDRs in each chainare held in their three-dimensional structure by the framework regionsand form together with the CDRs from the other chain the antigen bindingsite. The antibody's heavy and light chain CDR3 regions play aparticularly important role in the binding specificity/affinity of theantibodies according to the invention and therefore provide a furtherobject of the invention.

The term “antigen-binding portion of an antibody” when used herein referto the amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. “Framework” or “FR” regions are those variable domain regionsother than the hypervariable region residues as herein defined.Therefore, the light and heavy chain variable domains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding and defines the antibody'sproperties. CDR and FR regions are determined according to the standarddefinition of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991) and/or those residues from a “hypervariable loop”.

The terms “nucleic acid” or “nucleic acid molecule”, as used herein, areintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (tip, W), tyrosine (tyr, Y), and valine (val, V).

The antibodies according to the invention are preferably produced byrecombinant means. Therefore the antibody is preferably an isolatedmonoclonal antibody. Such recombinant methods are widely known in thestate of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody polypeptideand usually purification to a pharmaceutically acceptable purity. Forthe protein expression, nucleic acids encoding light and heavy chains orfragments thereof are inserted into expression vectors by standardmethods. Expression is performed in appropriate prokaryotic oreukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293cells, COS cells, yeast, or E. coli cells, and the antibody is recoveredfrom the cells (supernatant or cells after lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis, and others well known in the art. SeeAusubel, F., et al., ed. Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The monoclonal antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies are readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

The “Fc part” of an antibody is not involved directly in binding of anantibody to an antigen, but exhibit various effector functions. A “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Accordingto the heavy chain constant regions the different classes ofimmunoglobulins are called, and, respectively. The Fc part of anantibody is directly involved in ADCC (antibody-dependent cell-mediatedcytotoxicity) and CDC (complement-dependent cytotoxicity) based oncomplement activation, C1q binding and Fc receptor binding. Complementactivation (CDC) is initiated by binding of complement factor C1q to theFc part of most IgG antibody subclasses. While the influence of anantibody on the complement system is dependent on certain conditions,binding to C1q is caused by defined binding sites in the Fc part. Suchbinding sites are known in the state of the art and described e.g. byBoackle, R. J., et al., Nature 282 (1979) 742-743; Lukas, T. J., et al.,J. Immunol 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J. J., Mol.Immunol. 16 (1979) 907-917; Burton, D. R., et al., Nature 288 (1980)338-344; Thommesen, J. E., et al., Mol. Immunol 37 (2000) 995-1004;Idusogie, E. E., et al., J. Immuno1.164 (2000) 4178-4184; Hezareh, M.,et al., J. Virology 75 (2001) 12161-12168; Morgan, A., et al.,Immunology 86 (1995) 319-324; EP 0 307 434. Such binding sites are e.g.L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numberingaccording to EU index of Kabat, E. A., see below). Antibodies ofsubclass IgG1, IgG2 and IgG3 usually show complement activation and C1qand C3 binding, whereas IgG4 do not activate the complement system anddo not bind C1q and C3.

In one embodiment the antibody according to the invention comprises a Fcpart derived from human origin and preferably all other parts of thehuman constant regions. As used herein the term “Fc part derived fromhuman origin” denotes a Fc part which is either a Fc part of a humanantibody of the subclass IgG1, IgG2, IgG3 or IgG4, preferably a Fc partfrom human IgG1 subclass, a mutated Fc part from human IgG1 subclass(preferably with a mutation on L234A+L235A), a Fc part from human IgG4subclass or a mutated Fc part from human IgG4 subclass (preferably witha mutation on S228P). Mostly preferred are the human heavy chainconstant regions of SEQ ID NO: 58 (human IgG1 subclass), SEQ ID NO: 59(human IgG1 subclass with mutations L234A and L235A), SEQ ID NO: 60human IgG4 subclass), or SEQ ID NO: 61 (human IgG4 subclass withmutation S228P).

Preferably the antibody according to the invention is of human IgG1subclass or of human IgG4 subclass. In one embodiment the antibodyaccording to the invention is of human IgG1 subclass. In one embodimentthe antibody according to the invention is of human IgG4 subclass.

In one embodiment the antibody according to the invention ischaracterized in that the constant chains are of human origin. Suchconstant chains are well known in the state of the art and e.g.described by Kabat, E. A., (see e.g. Johnson, G. and Wu, T. T., NucleicAcids Res. 28 (2000) 214-218). For example, a useful human heavy chainconstant region comprises an amino acid sequence of SEQ ID NO: 58. Forexample, a useful human light chain constant region comprises an aminoacid sequence of a kappa-light chain constant region of SEQ ID NO: 57.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24, or    -   b) the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32, or    -   c) the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40, or    -   d) the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48, or    -   e) the heavy chain variable domain is SEQ ID NO:55 and the light        chain variable domain is SEQ ID NO:56.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24, or    -   b) the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32, or    -   c) the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40, or    -   d) the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:15 and the light        chain variable domain is SEQ ID NO:16, or a humanized version        thereof.

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:75 and the light        chain variable domain is SEQ ID NO:76;    -   or a humanized version thereof

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   the heavy chain variable domain is SEQ ID NO:83 and the light        chain variable domain is SEQ ID NO:84;    -   or a humanized version thereof

In one embodiment the antibody binding to human CSF-1R used in thecombination therapy is characterized in that

-   -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of        SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or,    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54; or    -   h) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region        of SEQ ID NO:71, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO:73, and        a CDR1 region of SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region        of SEQ ID NO: 79, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:80, a CDR2 region of SEQ ID NO: 81,        and a CDR1 region of SEQ ID NO: 82.

In one embodiment the combination therapy with an antibody binding tohuman CSF-1R, is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of        SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22.

In one embodiment the combination therapy with an antibody binding tohuman CSF-1R, is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of        SEQ ID NO: 27, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29, and        a CDR1 region of SEQ ID NO: 30.

In one embodiment the combination therapy with an antibody binding tohuman CSF-1R, is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of        SEQ ID NO: 35, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37, and        a CDR1 region of SEQ ID NO: 38.

In one embodiment the combination therapy with an antibody binding tohuman CSF-1R, is characterized in that

-   -   the heavy chain variable domain comprises a CDR3 region of SEQ        ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of        SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46.

The invention comprises a method for the treatment of a patient in needof therapy, characterized by administering to the patient atherapeutically effective amount of an antibody according to theinvention.

The invention comprises the use of an antibody according to theinvention for the described therapy.

One preferred embodiment of the invention are the CSF-1R antibodies ofthe present invention for use in the treatment of “CSF-1R mediateddiseases” or the CSF-1R antibodies of the present invention for use forthe manufacture of a medicament in the treatment of “CSF-1R mediateddiseases”, which can be described as follows:

There are 3 distinct mechanisms by which CSF-1R signaling is likelyinvolved in tumor growth and metastasis. The first is that expression ofCSF-ligand and receptor has been found in tumor cells originating in thefemale reproductive system (breast, ovarian, endometrium, cervical)(Scholl, S. M., et al., J. Natl. Cancer Inst. 86 (1994) 120-126;Kacinski, B. M., Mol. Reprod. Dev. 46 (1997) 71-74; Ngan, H. Y., et al.,Eur. J. Cancer 35 (1999) 1546-1550; Kirma, N., et al., Cancer Res 67(2007) 1918-1926) and the expression has been associated with breastcancer xenograft growth as well as poor prognosis in breast cancerpatients. Two point mutations were seen in CSF-1R in about 10-20% ofacute myelocytic leukemia, chronic myelocytic leukemia andmyelodysplasia patients tested in one study, and one of the mutationswas found to disrupt receptor turnover (Ridge, S. A., et al., Proc.Natl. Acad. Sci USA 87 (1990) 1377-1380). However the incidence of themutations could not be confirmed in later studies (Abu-Duhier, F. M., etal., Br. J. Haematol. 120 (2003) 464-470). Mutations were also found insome cases of hepatocellular cancer (Yang, D. H., et al., HepatobiliaryPancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis(Abu-Duhier, F. M., et al., Br. J. Haematol. 120 (2003) 464-470).Recently, in the GDM-1 cell line derived from a patient withmyelomonoblastic leukemia the Y571D mutation in CSF-1R was identified(Chase, A., et al., Leukemia 23 (2009) 358-364).

Pigmented villonodular synovitis (PVNS) and Tenosynovial Giant celltumors (TGCT) can occur as a result of a translocation that fuses theM-CSF gene to a collagen gene COL6A3 and results in overexpression ofM-CSF (West, R. B., et al., Proc. Natl. Acad. Sci. USA 103 (2006)690-695). A landscape effect is proposed to be responsible for theresulting tumor mass that consists of monocytic cells attracted by cellsthat express M-CSF. TGCTs are smaller tumors that can be relativelyeasily removed from fingers where they mostly occur. PVNS is moreaggressive as it can recur in large joints and is not as easilycontrolled surgically.

The second mechanism is based on blocking signaling through M-CSF/CSF-1Rat metastatic sites in bone which induces osteoclastogenesis, boneresorption and osteolytic bone lesions. Breast, multiple myeloma andlung cancers are examples of cancers that have been found to metastasizeto the bone and cause osteolytic bone disease resulting in skeletalcomplications. M-CSF released by tumor cells and stroma induces thedifferentiation of hematopoietic myeloid monocyte progenitors to matureosteoclasts in collaboration with the receptor activator of nuclearfactor kappa-B ligand-RANKL. During this process, M-CSF acts as apermissive factor by giving the survival signal to osteoclasts (Tanaka,S., et al., J. Clin. Invest. 91 (1993) 257-263) Inhibition of CSF-1Ractivity during osteoclast differentiation and maturation with ananti-CSF-1R antibody is likely to prevent unbalanced activity ofosteoclasts that cause osteolytic disease and the associated skeletalrelated events in metastatic disease. Whereas breast, lung cancer andmultiple myeloma typically result in osteolytic lesions, metastasis tothe bone in prostate cancer initially has an osteoblastic appearance inwhich increased bone forming activity results in ‘woven bone’ which isdifferent from typical lamellar structure of normal bone. During diseaseprogression bone lesions display a significant osteolytic component aswell as high serum levels of bone resorption and suggests thatanti-resorptive therapy may be useful. Bisphosphonates have been shownto inhibit the formation of osteolytic lesions and reduced the number ofskeletal-related events only in men with hormone-refractory metastaticprostate cancer but at this point their effect on osteoblastic lesionsis controversial and bisphosphonates have not been beneficial inpreventing bone metastasis or hormone responsive prostate cancer todate. The effect of anti-resorptive agents in mixedosteolytic/osteoblastic prostate cancer is still being studied in theclinic (Choueiri, M. B., et al., Cancer Metastasis Rev. 25 (2006)601-609; Vessella, R. L. and Corey, E., Clin. Cancer Res. 12 (20 Pt 2)(2006) 6285s-6290s).

The third mechanism is based on the recent observation that tumorassociated macrophages (TAM) found in solid tumors of the breast,prostate, ovarian and cervical cancers correlated with poor prognosis(Bingle, L., et al., J. Pathol. 196 (2002) 254-265; Pollard, J. W., Nat.Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to the tumor byM-CSF and other chemokines. The macrophages can then contribute to tumorprogression through the secretion of angiogenic factors, proteases andother growth factors and cytokines and may be blocked by inhibition ofCSF-1R signaling. Recently it was shown by Zins et al (Zins, K., et al.,Cancer Res. 67 (2007) 1038-1045) that expression of siRNA of Tumornecrosis factor alpha (TNF alpha), M-CSF or the combination of bothwould reduce tumor growth in a mouse xenograft model between 34% and 50%after intratumoral injection of the respective siRNA. SiRNA targetingthe TNF alpha secreted by the human SW620 cells reduced mouse M-CSFlevels and led to reduction of macrophages in the tumor. In additiontreatment of MCF7 tumor xenografts with an antigen binding fragmentdirected against M-CSF did result in 40% tumor growth inhibition,reversed the resistance to chemotherapeutics and improved survival ofthe mice when given in combination with chemotherapeutics (Paulus, P.,et al., Cancer Res. 66 (2006) 4349-4356).

TAMs are only one example of an emerging link between chronicinflammation and cancer. There is additional evidence for a link betweeninflammation and cancer as many chronic diseases are associated with anincreased risk of cancer, cancers arise at sites of chronicinflammation, chemical mediators of inflammation are found in manycancers; deletion of the cellular or chemical mediators of inflammationinhibits development of experimental cancers and long-term use ofanti-inflammatory agents reduce the risk of some cancers. A link tocancer exists for a number of inflammatory conditions among-those H.pylori induced gastritis for gastric cancer, Schistosomiasis for bladdercancer, HHVX for Kaposi's sarcoma, endometriosis for ovarian cancer andprostatitis for prostate cancer (Balkwill, F., et al., Cancer Cell 7(2005) 211-217). Macrophages are key cells in chronic inflammation andrespond differentially to their microenvironment. There are two types ofmacrophages that are considered extremes in a continuum of functionalstates: M1 macrophages are involved in Type 1 reactions. These reactionsinvolve the activation by microbial products and consequent killing ofpathogenic microorganisms that result in reactive oxygen intermediates.On the other end of the extreme are M2 macrophages involved in Type 2reactions that promote cell proliferation, tune inflammation andadaptive immunity and promote tissue remodeling, angiogenesis and repair(Mantovani, A., et al., Trends Immunol. 25 (2004) 677-686). Chronicinflammation resulting in established neoplasia is usually associatedwith M2 macrophages. A pivotal cytokine that mediates inflammatoryreactions is TNF alpha that true to its name can stimulate anti-tumorimmunity and hemorrhagic necrosis at high doses but has also recentlybeen found to be expressed by tumor cells and acting as a tumor promoter(Zins, K., et al., Cancer Res. 67 (2007) 1038-1045; Balkwill, F., CancerMetastasis Rev. 25 (2006) 409-416). The specific role of macrophageswith respect to the tumor still needs to be better understood includingthe potential spatial and temporal dependence on their function and therelevance to specific tumor types.

Thus one embodiment of the invention are the CSF-1R antibodies of thepresent invention for use in the treatment of cancer. The term “cancer”as used herein may be, for example, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma, lymphoma, lymphocytic leukemia, including refractoryversions of any of the above cancers, or a combination of one or more ofthe above cancers. In one preferred embodiment such cancer is a breastcancer, colorectal cancer, melanoma, head and neck cancer, lung canceror prostate cancer. In one preferred embodiment such cancer is a breastcancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer.In one preferred embodiment such cancers are further characterized byCSF-1 or CSF-1R expression or overexpression. One further embodiment theinvention are the CSF-1R antibodies of the present invention for use inthe simultaneous treatment of primary tumors and new metastases.

Thus another embodiment of the invention are the CSF-1R antibodies ofthe present invention for use in the treatment of periodontitis,histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone lossdue to cancer therapy, periprosthetic osteolysis, glucocorticoid-inducedosteoporosis, rheumatoid arthritis, psiratic arthritis, osteoarthritis,inflammatory arthridities, and inflammation.

Rabello, D., et al., Biochem. Biophys. Res. Commun. 347 (2006) 791-796has demonstrated that SNPs in the CSF1 gene exhibited a positiveassociation with aggressive periodontitis: an inflammatory disease ofthe periodontal tissues that causes tooth loss due to resorption of thealveolar bone.

Histiocytosis X (also called Langerhans cell histiocytosis, LCH) is aproliferative disease of Langerhans dendritic cells that appear todifferentiate into osteoclasts in bone and extra osseous LCH lesions.Langerhans cells are derived from circulating monocytes. Increasedlevels of M-CSF that have been measured in sera and lesions where foundto correlate with disease severity (da Costa, C. E., et al., J. Exp.Med. 201 (2005) 687-693). The disease occurs primarily in a pediatricpatient population and has to be treated with chemotherapy when thedisease becomes systemic or is recurrent.

The pathophysiology of osteoporosis is mediated by loss of bone formingosteoblasts and increased osteoclast dependent bone resorption.Supporting data has been described by Cenci et al showing that ananti-M-CSF antibody injection preserves bone density and inhibits boneresorption in ovariectomized mice (Cenci, S., et al., J. Clin. Invest.105 (2000) 1279-1287). Recently a potential link between postmenopausalbone loss due to estrogen deficiency was identified and found that thepresence of TNF alpha producing T-cell affected bone metabolism (Roggia,C., et al., Minerva Med. 95 (2004) 125-132). A possible mechanism couldbe the induction of M-CSF by TNF alpha in vivo. An important role forM-CSF in TNF-alpha-induced osteoclastogenesis was confirmed by theeffect of an antibody directed against M-CSF that blocked the TNF alphainduced osteolysis in mice and thereby making inhibitors of CSF-1Rsignaling potential targets for inflammatory arthritis (Kitaura, H., etal., J. Clin. Invest. 115 (2005) 3418-3427).

Paget's disease of bone (PDB) is the second most common bone metabolismdisorder after osteoporosis in which focal abnormalities of increasedbone turnover lead to complications such as bone pain, deformity,pathological fractures and deafness. Mutations in four genes have beenidentified that regulate normal osteoclast function and predisposeindividuals to PDB and related disorders: insertion mutations inTNFRSF11A, which encodes receptor activator of nuclear factor (NF)kappaB (RANK)-a critical regulator of osteoclast function, inactivatingmutations of TNFRSF11B which encodes osteoprotegerin (a decoy receptorfor RANK ligand), mutations of the sequestosome 1 gene (SQSTM1), whichencodes an important scaffold protein in the NFkappaB pathway andmutations in the valosin-containing protein (VCP) gene. This geneencodes VCP, which has a role in targeting the inhibitor of NFkappaB fordegradation by the proteasome (Daroszewska, A. and Ralston, S. H., Nat.Clin. Pract. Rheumatol. 2 (2006) 270-277). Targeted CSF-1R inhibitorsprovide an opportunity to block the deregulation of the RANKL signalingindirectly and add an additional treatment option to the currently usedbisphosphonates.

Cancer therapy induced bone loss especially in breast and prostatecancer patients is an additional indication where a targeted CSF-1Rinhibitor could prevent bone loss (Lester, J. E., et al., Br. J. Cancer94 (2006) 30-35). With the improved prognosis for early breast cancerthe long-term consequences of the adjuvant therapies become moreimportant as some of the therapies including chemotherapy, irradiation,aromatase inhibitors and ovary ablation affect bone metabolism bydecreasing the bone mineral density, resulting in increased risk forosteoporosis and associated fractures (Lester, J. E., et al., Br. J.Cancer 94 (2006) 30-35). The equivalent to adjuvant aromatase inhibitortherapy in breast cancer is androgen ablation therapy in prostate cancerwhich leads to loss of bone mineral density and significantly increasesthe risk of osteoporosis-related fractures (Stoch, S. A., et al., J.Clin. Endocrinol. Metab. 86 (2001) 2787-2791).

Targeted inhibition of CSF-1R signaling is likely to be beneficial inother indications as well when targeted cell types include osteoclastsand macrophages e.g. treatment of specific complications in response tojoint replacement as a consequence of rheumatoid arthritis. Implantfailure due to periprosthetic bone loss and consequent loosing ofprostheses is a major complication of joint replacement and requiresrepeated surgery with high socioeconomic burdens for the individualpatient and the health-care system. To date, there is no approved drugtherapy to prevent or inhibit periprosthetic osteolysis (Drees, P., etal., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).

Glucocorticoid-induced osteoporosis (GIOP) is another indication inwhich a CSF-1R inhibitor could prevent bone loss after longtermglucocorticocosteroid use that is given as a result of variousconditions among those chronic obstructive pulmonary disease, asthma andrheumatoid arthritis (Guzman-Clark, J. R., et al., Arthritis Rheum. 57(2007) 140-146; Feldstein, A. C., et al., Osteoporos. Int. 16 (2005)2168-2174).

Rheumatoid arthritis, psioratic arthritis and inflammatory arthriditiesare in itself potential indications for CSF-1R signaling inhibitors inthat they consist of a macrophage component and to a varying degree bonedestruction (Ritchlin, C. T., et al., J. Clin. Invest. 111 (2003)821-831). Osteoarthritis and rheumatoid arthritis are inflammatoryautoimmune disease caused by the accumulation of macrophages in theconnective tissue and infiltration of macrophages into the synovialfluid, which is at least partially mediated by M-CSF. Campbell, I., K.,et al., J. Leukoc. Biol. 68 (2000) 144-150, demonstrated that M-CSF isproduced by human jointtissue cells (chondrocytes, synovial fibroblasts)in vitro and is found in synovial fluid of patients with rheumatoidarthritis, suggesting that it contributes to the synovial tissueproliferation and macrophage infiltration which is associated with thepathogenesis of the disease. Inhibition of CSF-1R signaling is likely tocontrol the number of macrophages in the joint and alleviate the painfrom the associated bone destruction. In order to minimize adverseeffects and to further understand the impact of the CSF-1R signaling inthese indications, one method is to specifically inhibit CSF-1R withouttargeting a myriad other kinases, such as Raf kinase.

Recent literature reports correlate increased circulating M-CSF withpoor prognosis and atherosclerotic progression in chronic coronaryartery disease (Saitoh, T., et al., J. Am. Coll. Cardiol. 35 (2000)655-665; Ikonomidis, I., et al., Eur. Heart. J. 26 (2005) p. 1618-1624);M-CSF influences the atherosclerotic process by aiding the formation offoam cells (macrophages with ingested oxidized LDL) that express CSF-1Rand represent the initial plaque (Murayama, T., et al., Circulation 99(1999) 1740-1746).

Expression and signaling of M-CSF and CSF-1R is found in activatedmicroglia. Microglia, which are resident macrophages of the centralnervous system, can be activated by various insults, including infectionand traumatic injury. M-CSF is considered a key regulator ofinflammatory responses in the brain and M-CSF levels increase in HIV-1,encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis asa consequence of autocrine signaling by M-CSF/CSF-1R results ininduction of inflammatory cytokines and nitric oxides being released asdemonstrated by e.g. using an experimental neuronal damage model (Hao,A. J., et al., Neuroscience 112 (2002) 889-900; Murphy, G. M., Jr., etal., J. Biol. Chem. 273 (1998) 20967-20971). Microglia that haveincreased expression of CSF-1R are found to surround plaques in AD andin the amyloid precursor protein V717F transgenic mouse model of AD(Murphy, G. M., Jr., et al., Am. J. Pathol. 157 (2000) 895-904). On theother hand op/op mice with fewer microglia in the brain resulted infibrilar deposition of A-beta and neuronal loss compared to normalcontrol suggesting that microglia do have a neuroprotective function inthe development of AD lacking in the op/op mice (Kaku, M., et al., BrainRes. Brain Res. Protoc. 12 (2003) 104-108).

Expression and signaling of M-CSF and CSF-1R is associated withinflammatory bowel disease (IBD) (WO 2005/046657). The term“inflammatory bowel disease” refers to serious, chronic disorders of theintestinal tract characterized by chronic inflammation at various sitesin the gastrointestinal tract, and specifically includes ulcerativecolitis (UC) and Crohn's disease.

Thus another embodiment of the invention are the CSF-1R antibodies ofthe present invention for use in the treatment of periodontitis,histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone lossdue to cancer therapy, periprosthetic osteolysis, glucocorticoid-inducedosteoporosis, rheumatoid arthritis, psiratic arthritis, osteoarthritis,inflammatory arthridities, and inflammation.

The invention comprises the combination therapy with an antibody bindingto human CSF-1R being characterized by the above mentioned epitopebinding properties or alternatively by the above mentioned amino acidsequences and amino acid sequence fragments with an TLR9 agonist for thetreatment of cancer.

The invention comprises the combination therapy with an antibody bindingto human CSF-1R being characterized by the above mentioned epitopebinding properties or alternatively by the above mentioned amino acidsequences and amino acid sequence fragments with an TLR9 agonist for thetreatment of bone loss.

The invention comprises the combination therapy with an antibody bindingto human CSF-1R being characterized by the above mentioned epitopebinding properties or alternatively by the above mentioned amino acidsequences and amino acid sequence fragments with an TLR9 agonist for theprevention or treatment of metastasis.

The invention comprises the combination therapy with an antibody bindingto human CSF-1R being characterized by the above mentioned epitopebinding properties or alternatively by the above mentioned amino acidsequences and amino acid sequence fragments with an TLR9 agonist fortreatment of inflammatory diseases.

The invention comprises the use of an antibody characterized incomprising the antibody binding to human CSF-1R being characterized bythe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor the combination treatment of cancer as described herein oralternatively for the manufacture of a medicament for the combinationtreatment of cancer with an TLR9 agonist as described herein.

The invention comprises the use of an antibody characterized incomprising the antibody binding to human CSF-1R being characterized bythe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor the combination treatment as described herein of bone loss oralternatively for the manufacture of a medicament for the combinationtreatment of bone loss with an TLR9 agonist as described herein.

The invention comprises the use of an antibody characterized incomprising the antibody binding to human CSF-1R being characterized bythe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor the prevention or treatment of metastasis with the combination asdescribed herein or alternatively for the manufacture of a medicamentfor the prevention or treatment of metastasis with the combination withan TLR9 agonist as described herein.

The invention comprises the use of an antibody characterized incomprising the antibody binding to human CSF-1R being characterized bythe above mentioned epitope binding properties or alternatively by theabove mentioned amino acid sequences and amino acid sequence fragmentsfor combination treatment of inflammatory diseases as described hereinor alternatively for the manufacture of a medicament for the combinationtreatment of inflammatory diseases with an TLR9 agonist as describedherein.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COScells, yeast, or E. coli cells, and the antibody is recovered from thecells (from the supernatant or after cells lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified, or substantially pure form. Purification isperformed in order to eliminate other cellular components or othercontaminants, e.g. other cellular nucleic acids or proteins, by standardtechniques, including alkaline/SDS treatment, CsCl banding, columnchromatography, agarose gel electrophoresis, and others well known inthe art. See Ausubel, F., et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; Norderhaug, L., et al., J. Immunol Methods 204(1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30(1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

Nucleic acid molecules encoding amino acid sequence variants ofanti-CSF-1R antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of humanized anti-CSF-1Rantibody.

The heavy and light chain variable domains according to the inventionare combined with sequences of promoter, translation initiation,constant region, 3′ untranslated region, polyadenylation, andtranscription termination to form expression vector constructs. Theheavy and light chain expression constructs can be combined into asingle vector, co-transfected, serially transfected, or separatelytransfected into host cells which are then fused to form a single hostcell expressing both chains.

In another aspect, the present invention provides a composition, e.g. apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or the antigen-binding portion thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption/resorption delaying agents, and the likethat are physiologically compatible. Preferably, the carrier is suitablefor injection or infusion.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the preparation of sterileinjectable solutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. In addition towater, the carrier can be, for example, an isotonic buffered salinesolution.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient (effectiveamount). The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The term “a method of treating” or its equivalent, when applied to, forexample, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in a patient,or to alleviate the symptoms of a cancer. “A method of treating” canceror another proliferative disorder does not necessarily mean that thecancer cells or other disorder will, in fact, be eliminated, that thenumber of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of a patient, is nevertheless deemed toinduce an overall beneficial course of action.

The terms “administered in combination with” or “co-administration”,“co-administering” or “a combination” refer to the administration of theanti-CSF-1R, and the TLR 9 agonist e.g. as separateformulations/applications (or as one single formulation/application).The co-administration can be simultaneous or sequential in either order,wherein preferably there is a time period while both (or all) activeagents simultaneously exert their biological activities. Said antibodyand said TLR9 agonist are co-administered either simultaneously orsequentially (e.g. intravenous (i.v.) through a continuous infusion.When both therapeutic agents are co-administered sequentially the doseis administered either on the same day in two separate administrations,or one of the agents is administered on day 1 and the second isco-administered on day 2 to day 7, preferably on day 2 to 4. Thus in oneembodiment the term “sequentially” means within 7 days after the dose ofthe first component, preferably within 4 days after the dose of thefirst component; and the term “simultaneously” means at the same time.The terms “co-administration” with respect to the maintenance doses ofanti-CSF-1R antibody mean that the maintenance doses can be eitherco-administered simultaneously, if the treatment cycle is appropriatefor both drugs, e.g. every week. Or the further agent is e.g.administered e.g. every first to third day and said antibody isadministered every week. Or the maintenance doses are co-administeredsequentially, either within one or within several days.

It is self-evident that the antibodies are administered to the patientin a “therapeutically effective amount” (or simply “effective amount”)which is the amount of the respective compound or combination that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The amount of co-administration and the timing of co-administration willdepend on the type (species, gender, age, weight, etc.) and condition ofthe patient being treated and the severity of the disease or conditionbeing treated. Said anti-CSF-1R antibody and further agent are suitablyco-administered to the patient at one time or over a series oftreatments e.g. on the same day or on the day after.

Depending on the type and severity of the disease, about 0.1 mg/kg to 50mg/kg (e.g. 0.1-20 mg/kg) of said anti-CSF-1R antibody; is an initialcandidate dosage for co-administration of both drugs to the patient Theinvention comprises the use of the antibodies according to the inventionfor the treatment of a patient suffering from cancer, especially fromcolon, lung or pancreas cancer.

Depending on the type and severity of the disease, about 0.1 mg/kg to 50mg/kg (e.g. 0.1-20 mg/kg) of said anti-CSF-1R antibody; is an initialcandidate dosage for co-administration of both drugs to the patient Theinvention comprises the use of the antibodies according to the inventionfor the treatment of a patient suffering from cancer, especially fromcolon, lung or pancreas cancer.

In addition to the anti-CSF-1R antibody in combination with the TLR9agonist also a chemotherapeutic agent can be administered.

In one embodiment such additional chemotherapeutic agents, which may beadministered with anti-CSF-1R antibody and the TLR9 agonist, include,but are not limited to, anti-neoplastic agents including alkylatingagents including: nitrogen mustards, such as mechlorethamine,cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas,such as carmustine (BCNU), lomustine (CCNU), and semustine(methyl-CCNU); Temodal™ (temozolamide), ethylenimines/methylmelaminesuch as thriethylenemelamine (TEM), triethylene, thiophosphoramide(thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates suchas busulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-merca.rho.topurine,6-thioguamne, azathioprine, T-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; pipodophylotoxins such as etoposide andteniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycinC, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platinumcoordination complexes such as oxaliplatin, cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; Gemzar™ (gemcitabine), progestinsuch as hydroxyprogesterone caproate, medroxyprogesterone acetate andmegestrol acetate; estrogen such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogen such as tamoxifen; androgensincluding testosterone propionate and fluoxymesterone/equivalents;antiandrogens such as flutamide, gonadotropin-releasing hormone analogsand leuprolide; and non-steroidal antiandrogens such as flutamide.Therapies targeting epigenetic mechanism including, but not limited to,histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) andrelease of transcriptional repression (ATRA) therapies can also becombined with the antigen binding proteins. In one embodiment thechemotherapeutic agent is selected from the group consisting of taxanes(like e.g. paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel(e.g., Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib(Nexavar), and other multikinase inhibitors, oxaliplatin, cisplatin andcarboplatin, etoposide, gemcitabine, and vinblastine. In one embodimentthe chemotherapeutic agent is selected from the group consisting oftaxanes (like e.g. taxol (paclitaxel), docetaxel (Taxotere), modifiedpaclitaxel (e.g. Abraxane and Opaxio). In one embodiment, the additionalchemotherapeutic agent is selected from 5-fluorouracil (5-FU),leucovorin, irinotecan, or oxaliplatin. In one embodiment thechemotherapeutic agent is 5-fluorouracil, leucovorin and irinotecan(FOLFIRI). In one embodiment the chemotherapeutic agent is5-fluorouracil, and oxaliplatin (FOLFOX).

Specific examples of combination therapies with additionalchemotherapeutic agents include, for instance, therapies taxanes (e.g.,docetaxel or paclitaxel) or a modified paclitaxel (e.g., Abraxane orOpaxio), doxorubicin), capecitabine and/or bevacizumab (Avastin) for thetreatment of breast cancer; therapies with carboplatin, oxaliplatin,cisplatin, paclitaxel, doxorubicin (or modified doxorubicin (Caelyx orDoxil)), or topotecan (Hycamtin) for ovarian cancer, the therapies witha multi-kinase inhibitor, MKI, (Sutent, Nexavar, or 706) and/ordoxorubicin for treatment of kidney cancer; therapies with oxaliplatin,cisplatin and/or radiation for the treatment of squamous cell carcinoma;therapies with taxol and/or carboplatin for the treatment of lungcancer.

Therefore, in one embodiment the additional chemotherapeutic agent isselected from the group of taxanes (docetaxel or paclitaxel or amodified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabineand/or bevacizumab for the treatment of breast cancer.

In one embodiment the CSF-1R antibody/TLR9 agonist combination therapyis no chemotherapeutic agents are administered.

The invention comprises also a method for the treatment of a patientsuffering from such disease.

The invention further provides a method for the manufacture of apharmaceutical composition comprising an effective amount of an antibodyaccording to the invention together with a pharmaceutically acceptablecarrier and the use of the antibody according to the invention for sucha method.

The invention further provides the use of an antibody according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from cancer.

The invention also provides the use of an antibody according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from cancer.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Description of the Sequences

SEQ ID NO: 1 heavy chain CDR3, Mab 2F11SEQ ID NO: 2 heavy chain CDR2, Mab 2F11SEQ ID NO: 3 heavy chain CDR1, Mab 2F11SEQ ID NO: 4 light chain CDR3, Mab 2F11SEQ ID NO: 5 light chain CDR2, Mab 2F11SEQ ID NO: 6 light chain CDR1, Mab 2F11SEQ ID NO: 7 heavy chain variable domain, Mab 2F11SEQ ID NO: 8 light chain variable domain, Mab 2F11SEQ ID NO: 9 heavy chain CDR3, Mab 2E10SEQ ID NO: 10 heavy chain CDR2, Mab 2E10SEQ ID NO: 11 heavy chain CDR1, Mab 2E10SEQ ID NO: 12 light chain CDR3, Mab 2E10SEQ ID NO: 13 light chain CDR2, Mab 2E10SEQ ID NO: 14 light chain CDR1, Mab 2E10SEQ ID NO: 15 heavy chain variable domain, Mab 2E10SEQ ID NO: 16 light chain variable domain, Mab 2E10SEQ ID NO: 17 heavy chain CDR3, hMab 2F11-c11SEQ ID NO: 18 heavy chain CDR2, hMab 2F11-c11SEQ ID NO: 19 heavy chain CDR1, hMab 2F11-c11SEQ ID NO: 20 light chain CDR3, hMab 2F11-c11SEQ ID NO: 21 light chain CDR2, hMab 2F11-c11SEQ ID NO: 22 light chain CDR1, hMab 2F11-c11SEQ ID NO: 23 heavy chain variable domain, hMab 2F11-c11SEQ ID NO: 24 light chain variable domain, hMab 2F11-c11SEQ ID NO: 25 heavy chain CDR3, hMab 2F11-d8SEQ ID NO: 26 heavy chain CDR2, hMab 2F11-d8SEQ ID NO: 27 heavy chain CDR1, hMab 2F11-d8SEQ ID NO: 28 light chain CDR3, hMab 2F11-d8SEQ ID NO: 29 light chain CDR2, hMab 2F11-d8SEQ ID NO: 30 light chain CDR1, hMab 2F11-d8SEQ ID NO: 31 heavy chain variable domain, hMab 2F11-d8SEQ ID NO: 32 light chain variable domain, hMab 2F11-d8SEQ ID NO: 33 heavy chain CDR3, hMab 2F11-e7SEQ ID NO: 34 heavy chain CDR2, hMab 2F11-e7SEQ ID NO: 35 heavy chain CDR1, hMab 2F11-e7SEQ ID NO: 36 light chain CDR3, hMab 2F11-e7SEQ ID NO: 37 light chain CDR2, hMab 2F11-e7SEQ ID NO: 38 light chain CDR1, hMab 2F11-e7SEQ ID NO: 39 heavy chain variable domain, hMab 2F11-e7SEQ ID NO: 40 light chain variable domain, hMab 2F11-e7SEQ ID NO: 41 heavy chain CDR3, hMab 2F11-f12SEQ ID NO: 42 heavy chain CDR2, hMab 2F11-f12SEQ ID NO: 43 heavy chain CDR1, hMab 2F11-f12SEQ ID NO: 44 light chain CDR3, hMab 2F11-f12SEQ ID NO: 45 light chain CDR2, hMab 2F11-f12SEQ ID NO: 46 light chain CDR1, hMab 2F11-f12SEQ ID NO: 47 heavy chain variable domain, hMab 2F11-f12SEQ ID NO: 48 light chain variable domain, hMab 2F11-f12SEQ ID NO: 49 heavy chain CDR3, hMab 2F11-g1SEQ ID NO: 50 heavy chain CDR2, hMab 2F11-g1SEQ ID NO: 51 heavy chain CDR1, hMab 2F11-g1SEQ ID NO: 52 light chain CDR3, hMab 2F11-g1SEQ ID NO: 53 light chain CDR2, hMab 2F11-g1SEQ ID NO: 54 light chain CDR1, hMab 2F11-g1SEQ ID NO: 55 heavy chain variable domain, hMab 2F11-g1SEQ ID NO: 56 light chain variable domain, hMab 2F11-g1SEQ ID NO: 57 human kappa light chain constant regionSEQ ID NO: 58 human heavy chain constant region derived from IgG1SEQ ID NO: 59 human heavy chain constant region derived from IgG1mutated on L234A and L235ASEQ ID NO: 60 human heavy chain constant region derived from IgG4SEQ ID NO: 61 human heavy chain constant region derived from IgG4mutated on S228PSEQ ID NO: 62 human wildtype CSF-1R (wt CSF-1R)SEQ ID NO: 63 human mutant CSF-1R L301S Y969FSEQ ID NO: 64 human CSF-1R Extracellular Domain (domains D1-D5)SEQ ID NO: 65 human CSF-1R fragment delD4SEQ ID NO: 66 human CSF-1R fragment domains D1-D3SEQ ID NO: 67 signal peptide

SEQ ID NO: 68 Primer

SEQ ID NO: 69 heavy chain CDR3, Mab 1G10SEQ ID NO: 70 heavy chain CDR2, Mab 1G10SEQ ID NO: 71 heavy chain CDR1, Mab 1G10SEQ ID NO: 72 light chain CDR3, Mab 1G10SEQ ID NO: 73 light chain CDR2, Mab 1G10SEQ ID NO: 74 light chain CDR1, Mab 1G10SEQ ID NO: 75 heavy chain variable domain, Mab 1G10SEQ ID NO: 76 light chain variable domain, Mab 1G10SEQ ID NO: 77 heavy chain CDR3, Mab 2H7SEQ ID NO: 78 heavy chain CDR2, Mab 2H7SEQ ID NO: 79 heavy chain CDR1, Mab 2H7SEQ ID NO: 80 light chain CDR3, Mab 2H7SEQ ID NO: 81 light chain CDR2, Mab 2H7SEQ ID NO: 82 light chain CDR1, Mab 2H7SEQ ID NO: 83 heavy chain variable domain, Mab 2H7SEQ ID NO: 84 light chain variable domain, Mab 2H7SEQ ID NO: 85 human CSF-1R fragment domains D4-D5SEQ ID NO: 86 human CSF-1SEQ ID NO: 87 human IL-34SEQ ID NO: 88 human toll-like receptor 9 (TLR9)SEQ ID NO: 89 TLR9 agonist CpG ODN 2216SEQ ID NO: 90 TLR9 agonist CpG ODN PB4SEQ ID NO: 91 TLR9 agonist CpG ODN 1002SEQ ID NO: 92 TLR9 agonist CpG-7909 (CpG 2006, PF-3512676, Agatolimod)SEQ ID NO: 93 TLR9 agonist CpG-685 (GNKG168; CpG ODN; SBI Biotech)SEQ ID NO: 94 TLR9 agonist CpG-684SEQ ID NO: 95 TLR9 agonist CpG-28

In the following embodiment of the invention are described:

-   1. An antibody which binds to human CSF-1R wherein the antibody is    administered in combination with a Toll-like receptor 9 (TLR9)    agonist for use in the treatment of cancer.-   2. Use of a combination of    -   i) an antibody which binds to human CSF-1R, and    -   ii) a Toll-like receptor 9 (TLR9) agonist    -   for the manufacture of a medicament for use in the treatment of        cancer.-   3. The antibody or use according to embodiments 1 or 2, wherein the    cancer is further characterized by CSF-1R expression or    overexpression.-   4. The antibody or use according to any one of embodiments 1 or 2,    wherein the cancer is a breast cancer, colorectal cancer, melanoma,    head and neck cancer, lung cancer or prostate cancer.-   5. An antibody which binds to human CSF-1R characterized in binding    to the (dimerization) domains D4 to D5 (SEQ ID No: 85) of the    extracellular domain of human CSF-1R for use in    -   a) the inhibition of cell proliferation in CSF-1R        ligand-dependent and/or CSF-1 ligand-independent CSF-1R        expressing tumor cells;    -   b) the inhibition of cell proliferation of tumors with CSF-1R        ligand-dependent and/or CSF-1R ligand-independent CSF-1R        expressing macrophage infiltrate;    -   c) the inhibition of cell survival (in CSF-1R ligand-dependant        and/or CSF-1R ligand-independent) CSF-1R expressing monocytes        and macrophages; and/or    -   d) the inhibition of cell differentiation (in CSF-1R        ligand-dependent and/or CSF-1R ligand-independent) CSF-1R        expressing monocytes into macrophages,    -   wherein the antibody is administered in combination with a TLR9        agonist.-   6. Use of a combination of    -   i) an antibody which binds to the (dimerization) domains D4 to        D5 (SEQ ID No: 85) of the extracellular domain of human CSF-1R,        and    -   ii) a Toll-like receptor 9 (TLR9) agonist    -   for the manufacture of a medicament for use in        -   a) the inhibition of cell proliferation in CSF-1R            ligand-dependent and/or CSF-1 ligand-independent CSF-1R            expressing tumor cells;        -   b) the inhibition of cell proliferation of tumors with            CSF-1R ligand-dependent and/or CSF-1R ligand-independent            CSF-1R expressing macrophage infiltrate;        -   c) the inhibition of cell survival (in CSF-1R            ligand-dependant and/or CSF-1R ligand-independent) CSF-1R            expressing monocytes and macrophages; and/or        -   d) the inhibition of cell differentiation (in CSF-1R            ligand-dependent and/or CSF-1R ligand-independent) CSF-1R            expressing monocytes into macrophages.-   7. An antibody which binds to human CSF-1R, for use in the treatment    of a patient having a CSF-1R expressing tumor or having a tumor with    CSF-1R expressing macrophage infiltrate, wherein the tumor is    characterized by an increase of CSF-1R ligand and wherein the    anti-CSF-1R antibody is administered in combination with a TLR9    agonist.-   8. Use of a combination of    -   i) an antibody which binds to human CSF-1R, and    -   ii) a Toll-like receptor 9 (TLR9) agonist    -   for the manufacture of a medicament for use in for use in the        treatment of a patient having a CSF-1R expressing tumor or        having a tumor with CSF-1R expressing macrophage infiltrate,        wherein the tumor is characterized by an increase of CSF-1R        ligand.-   9. The antibody or use according to any one of embodiments 1 or 8    wherein the TLR9 agonist is characterized by induction of IFN-alpha,    IL-6, and/or IL-12 in plasmacytoid dendritic cells (pDCs).-   10. The antibody or use according to any one of embodiments 1 to 8,    wherein the TLR9 agonist is is a oligodeoxynucleotides containing    cytosine-phosphate-guanosine (CpG) motifs (CpG ODNs).-   11. The antibody or use according to any one of embodiments 1 to 10    wherein the antibody is characterized in binding to the domains D4    to D5 (SEQ ID No: 85) of the extracellular domain of human CSF-1R.-   12. The antibody according any one of the preceding embodiments,    wherein the antibody is characterized in that the antibody does not    bind to human CSF-1R fragment delD4 (SEQ ID NO: 65).-   13. The antibody according any one of the preceding embodiments,    characterized in that    -   a) the heavy chain variable domain is SEQ ID NO:7 and the light        chain variable domain is SEQ ID NO:8,    -   b) the heavy chain variable domain is SEQ ID NO:15 and the light        chain variable domain is SEQ ID NO:16;    -   c) the heavy chain variable domain is SEQ ID NO:75 and the light        chain variable domain is SEQ ID NO:76;    -   d) the heavy chain variable domain is SEQ ID NO:83 and the light        chain variable domain is SEQ ID NO:84;    -   e) the heavy chain variable domain is SEQ ID NO:23 and the light        chain variable domain is SEQ ID NO:24, or    -   f) the heavy chain variable domain is SEQ ID NO:31 and the light        chain variable domain is SEQ ID NO:32, or    -   g) the heavy chain variable domain is SEQ ID NO:39 and the light        chain variable domain is SEQ ID NO:40, or    -   h) the heavy chain variable domain is SEQ ID NO:47 and the light        chain variable domain is SEQ ID NO:48, or    -   i) the heavy chain variable domain is SEQ ID NO:55 and the light        chain variable domain is SEQ ID NO:56.-   14. The antibody according any one of the preceding embodiments,    characterized in that    -   a) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region        of SEQ ID NO:3, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a        CDR1 region of SEQ ID NO:6, or    -   b) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region        of SEQ ID NO: 11, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13,        and a CDR1 region of SEQ ID NO: 14, or    -   c) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region        of SEQ ID NO:19, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and        a CDR1 region of SEQ ID NO:22, or    -   d) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region        of SEQ ID NO: 27, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29,        and a CDR1 region of SEQ ID NO: 30, or    -   e) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region        of SEQ ID NO: 35, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37,        and a CDR1 region of SEQ ID NO: 38, or    -   f) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region        of SEQ ID NO:43, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and        a CDR1 region of SEQ ID NO:46, or    -   g) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region        of SEQ ID NO: 51, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53,        and a CDR1 region of SEQ ID NO: 54; or    -   h) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region        of SEQ ID NO:71, and the light chain variable domain comprises a        CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO:73, and        a CDR1 region of SEQ ID NO:74, or    -   i) the heavy chain variable domain comprises a CDR3 region of        SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region        of SEQ ID NO: 79, and the light chain variable domain comprises        a CDR3 region of SEQ ID NO:80, a CDR2 region of SEQ ID NO: 81,        and a CDR1 region of SEQ ID NO: 82.-   15. The antibody according any one of the preceding embodiments,    characterized in that said antibody is of human IgG1 subclass or is    of human IgG4 subclass.-   16. A method of treatment comprising administering to a patient    suffering from cancer an effective amount of an antibody which binds    to human CSF-1R wherein the antibody is administered in combination    with a Toll-like receptor 9 (TLR9) agonist.-   17. The method according to embodiment 16, wherein the cancer is    further characterized by CSF-1R expression or overexpression.-   18. The method according to embodiment 16, wherein the cancer is a    breast cancer, colorectal cancer, melanoma, head and neck cancer,    lung cancer or prostate cancer.-   19. A method comprising administering an effective amount of an    antibody which binds to human CSF-1R and is characterized in binding    to the (dimerization) domains D4 to D5 (SEQ ID No: 85) of the    extracellular domain of human CSF-1R for use in    -   a) the inhibition of cell proliferation in CSF-1R        ligand-dependent and/or CSF-1 ligand-independent CSF-1R        expressing tumor cells;    -   b) the inhibition of cell proliferation of tumors with CSF-1R        ligand-dependent and/or CSF-1R ligand-independent CSF-1R        expressing macrophage infiltrate;    -   c) the inhibition of cell survival (in CSF-1R ligand-dependant        and/or CSF-1R ligand-independent) CSF-1R expressing monocytes        and macrophages; and/or    -   d) the inhibition of cell differentiation (in CSF-1R        ligand-dependent and/or CSF-1R ligand-independent) CSF-1R        expressing monocytes into macrophages,    -   wherein the antibody is administered in combination with an        effective amount of a TLR9 agonist.-   20. A method of treatment comprising administering an effective    amount of an antibody which binds to human CSF-1R, for use in the    treatment of a patient having a CSF-1R expressing tumor or having a    tumor with CSF-1R expressing macrophage infiltrate, wherein the    tumor is characterized by an increase of CSF-1R ligand and wherein    the anti-CSF-1R antibody is administered in combination with an    effective amount of a TLR9 agonist.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

EXAMPLES Example 1 Generation of a Hybridoma Cell Line ProducingAnti-CSF-1R Antibodies

Immunization procedure of NMRI mice

NMRI mice were immunized with an expression vector pDisplay™(Invitrogen, USA) encoding the extracellular domain of huCSF-1R byutilizing electroporation. Every mouse was 4 times immunized with 100 μgDNA. When serum titers of anti-huCSF-1R were found to be sufficient,mice were additionally boosted once with 50 μg of a 1:1 mixture huCSF-1RECD/huCSF-1R ECDhuFc chimera in 200 μl PBS intravenously (i.v.) 4 and 3days before fusion.

Antigen specific ELISA

Anti-CSF-1R titers in sera of immunized mice were determined by antigenspecific ELISA.

0.3 μg/ml huCSF-1R-huFc chimera (soluble extracellular domain) wascaptured on a streptavidin plate (MaxiSorb; MicroCoat, DE, Cat. No.11974998/MC1099) with 0.1 mg/ml biotinylated anti Fcγ (JacksonImmunoResearch., Cat. No. 109-066-098) and horse radish peroxidase(HRP)-conjugated F(ab′)₂ anti-mouse IgG (GE Healthcare, UK, Cat. No.NA9310V) diluted 1/800 in PBS/0.05% Tween20/0.5% BSA was added. Serafrom all taps were diluted 1/40 in PBS/0.05% Tween20/0.5% BSA andserially diluted up to 1/1638400. Diluted sera were added to the wells.Pre-tap serum was used as negative control. A dilution series of mouseanti-human CSF-1R Mab3291 (R&D Systems, UK) from 500 ng/ml to 0.25 ng/mlwas used as positive control. All components were incubated together for1.5 hours, Wells were washed 6 times with PBST (PBS/0.2% Tween20) andassays were developed with freshly prepared ABTS® solution (1 mg/ml)(ABTS: 2,2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) for 10minutes at RT. Absorbance was measured at 405 nm.

Hybridoma Generation

The mouse lymphocytes can be isolated and fused with a mouse myelomacell line using PEG based standard protocols to generate hybridomas. Theresulting hybridomas are then screened for the production ofantigen-specific antibodies. For example, single cell suspensions ofsplenic derived lymphocytes from immunized mice are fused to Ag8non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) with 50%PEG. Cells are plated at approximately 10⁴ in flat bottom 96 well microtiter plate, followed by about two weeks incubation in selective medium.Individual wells are then screened by ELISA for human anti-CSF-1Rmonoclonal IgM and IgG antibodies. Once extensive hybridoma growthoccurs, the antibody secreting hybridomas are replated, screened again,and if still positive for human IgG, anti-CSF-1R monoclonal antibodies,can be subcloned by FACS. The stable subclones are then cultured invitro to produce antibody in tissue culture medium for characterization.Antibodies according to the invention could be selected using thedetermination of the binding of anti-CSF-1R antibodies to human CSF-1Rfragment delD4 and to human CSF-1R Extracellular Domain (CSF-1R-ECD) asdescribed in Example 4, as well as the determination of growthinhibition of NIH3T3 cells transfected with wildtype CSF-1R (liganddependent signalling) or mutant CSF-1R L301S Y969F (ligand independentsignalling) under treatment with anti-CSF-1R monoclonal antibodies asdescribed in Example 5.

Culture of Hybridomas

Generated muMAb hybridomas were cultured in RPMI 1640 (PAN—Catalogue No.(Cat. No.) PO4-17500) supplemented with 2 mM L-glutamine (GIBCO—Cat. No.35050-038), 1 mM Na-Pyruvat (GIBCO—Cat. No. 11360-039), 1×NEAA(GIBCO—Cat. No. 11140-035), 10% FCS (PAA—Cat. No. A15-649), 1× Pen Strep(Roche—Cat. No. 1074440), 1× Nutridoma CS (Roche—Cat. No. 1363743), 50μM Mercaptoethanol (GIBCO—Cat. No. 31350-010) and 50 Um′ IL 6 mouse(Roche—Cat. No. 1 444 581) at 37° C. and 5% CO₂. Some of the resultingmouse antibodies have been humanized (e.g. Mab 2F11) and been expressedrecombinantly.

Example 2 Inhibition of CSF-1 Binding to CSF-1R (ELISA)

By setting-up this assay to first allow for anti-CSF-1R antibody bindingto the CSF-1R-ECD followed by detection of ligand not bound to thereceptor both-ligand displacing antibodies and dimerization inhibitoranti-CSF-1R antibodies—can be tested. The test was performed on 384 wellmicrotiter plates (MicroCoat, DE, Cat. No. 464718) at RT. After eachincubation step plates were washed 3 times with PBST.

At the beginning, plates were coated with 0.5 mg/ml goat F(ab′)2biotinylated anti Fcγ (Jackson ImmunoResearch., Cat. No. 109-006-170)for 1 hour (h).

Thereafter the wells were blocked with PBS supplemented with 0.2%Tween®-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 0.5 h. 75 ng/ml ofhuCSF-1R-huFc chimera (which forms the dimeric soluble extracellulardomain of huCSF-1R) was immobilized to plate for 1 h. Then dilutions ofpurified antibodies in PBS/0.05% Tween20/0.5% BSA were incubated for 1h. After adding a mixture of 3 ng/ml hu CSF-1 (active 149 aa fragment ofhuman CSF-1 (aa 33-181 of SEQ ID NO: 86); Biomol, DE, Cat. No. 60530),50 ng/ml biotinylated anti CSF-1 clone BAF216 (R&D Systems, UK) and1:5000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat. No.11089153001) for 1 h the plates were washed 6 times with PBST. AntiCSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US), which inhibits theligand-receptor interaction, was used as positive control. Plates weredeveloped with freshly prepared BM Blue® POD substrate solution (BMBlue®: 3,3′-5,5′-Tetramethylbenzidine, Roche Diagnostics GmbH, DE, Cat.No. 11484281001) for 30 minutes at RT. Absorbance was measured at 370nm. A decrease of absorbance is found, if the anti-CSF-1R antibodycauses a release of CSF-1 from the dimeric complex. All anti-CSF-1Rantibodies showed significant inhibition of the CSF-1 interaction withCSF-1R (see Table 1). Anti CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, USsee also Sherr, C. J. et al., Blood 73 (1989) 1786-1793), which inhibitsthe ligand-receptor interaction, was used as reference control.

TABLE 1 Calculated IC50 values for the inhibition of the CSF-1/CSF-1Rinteraction IC50 CSF-1/CSF-1R Inhibition CSF-1R Mab [ng/ml] Mab 2F1119.3 Mab 2E10 20.6 Mab 2H7 18.2 Mab 1G10 11.8 SC-2-4A5 35.2

Example 3 Inhibition of CSF-1-Induced CSF-1R Phosphorylation inNIH3T3-CSF-1R Recombinant Cells

4.5×10³ NIH 3T3 cells, retrovirally infected with an expression vectorfor full-length CSF-1R, were cultured in DMEM (PAA Cat. No. E15-011), 2mM L-glutamine (Sigma, Cat. No. G7513, 2 mM Sodium pyruvate, 1×nonessential aminoacids, 10% FKS (PAA, Cat. No. A15-649) and 100 ng/mlPenStrep (Sigma, Cat. No. P4333 [10 mg/ml]) until they reachedconfluency. Thereafter cells were washed with serum-free DMEM media (PAACat. No. E15-011) supplemented with sodium selenite [5 ng/ml] (Sigma,Cat. No. S9133), transferrin [10 μg/ml] (Sigma, Cat. No. T8158), BSA[400 ng/ml] (Roche Diagnostics GmbH, Cat. No. 10735078), 4 mML-glutamine (Sigma, Cat. No. G7513), 2 mM sodium pyruvate (Gibco, Cat.No. 11360), 1× nonessential aminoacids (Gibco, Cat: 11140-035),2-mercaptoethanol [0.05 mM] (Merck, Cat. No. M7522), 100 ng/ml andPenStrep (Sigma, Cat. No. P4333) and incubated in 30 μl of the samemedium for 16 hours to allow for receptor up-regulation. 10 μl ofdiluted anti-CSR-1R antibodies were added to the cells for 1.5 h. Thencells were stimulated with 10 μl of 100 ng/ml hu CSF-1 (active 149 aafragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86); Biomol, DE, Cat.No. 60530) for 5 min. After the incubation, supernatant was removed,cells were washed twice with 80 μl of ice-cold PBS and 50 μl of freshlyprepared ice-cold lysis buffer (150 mM NaCl/20 mM Tris pH 7.5/1 mMEDTA/1 mM EGTA/1% Triton X-100/1 protease inhibitor tablet (RocheDiagnostics GmbH Cat. No. 1 836 170) per 10 ml buffer/10 μl/mlphosphatase inhibitor cocktail 1 (Sigma Cat. No. P-2850, 100× Stock)/10μl/ml protease inhibitor 1 (Sigma Cat. No. P-5726, 100× Stock)/10 μl/ml1 M NaF) was added. After 30 minutes on ice the plates were shakenvigourously on a plateshaker for 3 minutes and then centrifuged 10minutes at 2200 rpm (Heraeus Megafuge 10).

The presence of phosphorylated and total CSF-1 receptor in the celllysate was analyzed with Elisa. For detection of the phosphorylatedreceptor the kit from R&D Systems (Cat. No. DYC3268-2) was usedaccording to the instructions of the supplier. For detection of totalCSF-1R 10 μl of the lysate was immobilized on plate by use of thecapture antibody contained in the kit. Thereafter 1:750 dilutedbiotinylated anti CSF-1R antibody BAF329 (R&D Systems) and 1:1000diluted streptavidin-HRP conjugate was added. After 60 minutes plateswere developed with freshly prepared ABTS® solution and the absorbancewas detected. Data were calculated as % of positive control withoutantibody and the ratio value phospho/total receptor expressed. Thenegative control was defined without addition of M-CSF-1. Anti CSF-1R SC2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C. J. et al., Blood73 (1989) 1786-1793), which inhibits the ligand-receptor interaction,was used as reference control.

TABLE 2 Calculated IC50 values for the inhibition of CSF-1 receptorphosphorylation. IC50 CSF-1R Phosphorylation CSF-1R Mab [ng/ml] Mab 2F11219.4 Mab 2E10 752.0 Mab 2H7 703.4 Mab 1G10 56.6 SC-2-4A5 1006.6

Example 4 Determination of the Binding of Anti-CSF-1R Antibodies toHuman CSF-1R Fragment delD4 and to Human CSF-1R Extracellular Domain(CSF-1R-ECD)

Preparation of Human CSF-1R Extracellular Domain (CSF-1R-ECD)(Comprising the Extracellular Subdomains D1-D5, hCSF-1R-ECD) of SEQ IDNO: 64:

pCMV-preS-Fc-hCSF-1R-ECD (7836 bp) encodes the complete ECD of humanCSF-1R (SEQ ID NO: 64) C-terminally fused to a PreScission proteasecleavage site, followed by aa100-330 of human IgG1 and a 6×His-Tag,under the control of CMV promoter. The natural signal peptide has beenvaried by insertion of amino acids G and S after the first M, in orderto create a BamHI restriction site.

Preparation of Human CSF-1R Fragment delD4 (Comprising the ExtracellularSubdomains D1-D3 and D5, hCSF-1R-delD4) of SEQ ID NO: 65:

hCSF1R-delD4-V1-PreSc-hFc-His was cloned from pCMV-preS-Fc-hCSF-1R-ECDby means of the Stratagene QuikChange XL site-directed mutagenesisprotocol, using delD4-for with sequenceCACCTCCATGTTCTTCCGGTACCCCCCAGAGGTAAG (SEQ ID NO: 68) as the forwardprimer and delD4-rev with the reverse complement sequence as the reverseprimer. A protocol variation published in BioTechniques 26 (1999) 680was used to extend both primers in separate reactions in three cyclespreceeding the regular Stratagene protocol:

Two separate 50 μl reaction mixtures were set up according to themanufacturer's manual, each containing 10 ng plasmidpCMV-preS-Fc-hCSF1R-ECD as the template and 10 pM of one of the primersdelD4-for or delD4-rev, and 0.5 μl Pfu DNA polymerase as provided withthe kit. Three PCR cycles 95° C. 30 sec/55° C. 60 sec/68° C. 8 min wererun, then 25 μl each of both reaction mixtures were combined in a newtube and 0.5 μl fresh Pfu DNA polymerase were added. The regular PCRprotocol with 18 temperature cycles as specified by Stratagene in thekit manual was carried out, followed by 2 hrs final digestion with theDpn1 restriction enzyme provided with the kit. Clones bearing thedeletion were detected by digestion with Cel II and Not I and verifiedby sequencing.

Protein was prepared by transient transfection in the Hek293 FreeStylesuspension cell system (Invitrogen) according to the manufacturer'sspecifications. After 1 week 500 ml supernatant was filtered and loadedonto a 1 ml HiTrap MabSelect Xtra (GE healthcare) protein A column (0.2ml/min) The column was washed first with PBS, then with 50 mM Tris/150mM NaCl/1 mM EDTA/pH 7.3. 75 μl PreScission Protease (GE #27-0843-01)diluted in 375 μl of the same buffer were loaded onto the column and theclosed column was incubated over night at 4° C. with rolling. The columnwas mounted on top of a 1 ml GSTrap FF column (GE helthcare) and thedesired protein was eluted (0.2 ml/min, 0.2 ml fractions). Pooledfractions were concentrated from 1.8 ml to 0.4 ml by centrifugalultrafiltration via a 3 k Nanosep and chromatographed over an S200 HRSEC in PBS (0.5 ml/min).

Human CSF-1R fragment delD4 was obtained in two fractions as a dimericmolecule (pooll, V=1.5 ml; c=0.30 mg/ml; apparent mass on SDS page 83kDa, reduced 62 kDa) and as the monomer (pool 2, V=1.4 ml; c=0.25 mg/mlapparent mass on SDS page 62 kDa). The dimeric form was used for allexperiments.

Determination of the Binding of Anti-CSF-1R Antibodies to Human CSF-1RFragment delD4 and to Human CSF-1R Extracellular Domain (CSF-1R-ECD)(Binding Signals as Response Units (RU):

Instrument: Biacore T100 (GE Healthcare)

-   -   Software: T100 Control, Version 2.0.1        -   T100 Evaluation, Version 2.0.2

Assayformat Chip: CMS Temperature: 25° C.

CSF-1R fragments were immobilized via amine coupling. To compare thebinding of different anti-CSF-1R antibodies according to the inventionone concentration of the test antibody was injected. Anti CSF-1R Mab3291(R&D-Systems) and SC 2-4A5 (Santa Cruz Biotechnology, US— see alsoSherr, C. J. et al., Blood 73 (1989) 1786-1793), was used as referencecontrol, anti-CCR5 m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on18.08.2004 at DSMZ) as negative control, all under the same conditionsas the anti-CSF-1R antibodies according to the invention.

Amine Coupling of CSF-1R Fragments

Standard amine coupling according to the manufacturer's instructions:running buffer: PBS-T (Roche: 11 666 789+0.05% Tween20: 11 332 465),activation by mixture of EDC/NHS, injection of human CSF-1R fragmentdelD4 (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO:65) and human CSF-1R Extracellular Domain (CSF-1R-ECD) (comprising theextracellular subdomains D1-D5) (SEQ ID NO: 64) for 600 seconds at flowrate 10 μl/min; diluted in coupling buffer NaAc, pH 5.0, c=10 μg/mL;finally remaining activated carboxyl groups were blocked by injection of1 M Ethanolamin.

Binding of <CSF-1R>Mab 2F11, Mab 2E10, Mab 3291 and sc2-4A5 and OtherAnti-CSF-1R Antibodies to Human CSF-1R Fragment delD4 and Human CSF-1RExtracellular Domain (CSF-1R-ECD) at 25° C.

Running Buffer: PBS-T (Roche: 11 666 789+0.05% Tween20: 11 332 465)Analyte Sample:

Binding was measured at a flow rate of 30 μL/min by one injection of theanalyte with concentration c=10 nM. (for Mab 1G10, Mab 2H7 and humanizedhMab 2F11-e7 in second experiment) Each injection was 700 seconds long,followed by a dissociation phase of 180 seconds. Final regeneration wasperformed after each cycle using 50 mM NaOH, contact time 60 seconds,flow rate 30 μL/min.

Signals were measured by a report point 10 seconds after end ofinjection. Reference signals (signals from a blank reference flow cell(treated with EDC/NHS and ethanolamine, only) were subtracted to givethe binding signals (as RU). If binding signals of nonbinding antibodieswere slightly below 0 (Mab 2F11=−3; Mab 2E10=−2; Mab 1G10=−6, Mab2H7=−9; and humanized hMab 2F11-e7=−7) the values were set as 0.

TABLE 3a Binding of <CSF-1R> MAbs to human CSF-1R fragment delD4 andCSF-1R-ECD and ratio at 25° C., measured by SPR Binding Ratio of bindingof anti- Binding to CSF- CSF1R antibodies to to delD4 1R-ECD CSF1Rfragment delD4/ [RU] [RU] to CSF-1R-ECD Mab 3291 1015 627 1015/627 =1.61  sc2-4A5 374 249  374/249 = 1.50 Mab 2F11 0 176 0/176 = 0 hMab2F11-e7 0 237 0/237 = 0 Mab 2E10 0 120 0/120 = 0 Mab 1G10 0 2708 0/2708= 0  Mab 2H7 0 147 0/147 = 0 m<CCR5>Pz03.1C5 2 5 —

Mab 2F11 and Mab 2E10 showed binding to the human CSF-1R ExtracellularDomain (CSF-1R-ECD) (see FIG. 3b ); however no binding was detected toCSF-1R fragment delD4. (see FIG. 3a ).

Sc2-4A5 and MAB3291 showed binding to CSF-1R-ECD and to del D4 (seeFIGS. 3b and 3a ).

Thus the ratio of binding of anti-CSF1R antibodies Mab 2F11 and Mab 2E10to CSF1R fragment delD4/to CSF-1R-ECD was clearly below 1:50 (=0.02),while the binding ratio of MAB3291 and Sc2-4A5 were 1.61 and 1.50,respectively and were highly above 1:50 (=0.02). Negative controlantibody m<CCR5>Pz03.1C5 did not show any binding (as expected).

Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 showed binding to the humanCSF-1R Extracellular Domain (CSF-1R-ECD) (see FIG. 3d ); however nobinding was detected to CSF-1R fragment delD4. (see FIG. 3). Thus theratio of binding of anti-CSF1R antibodies Mab 1G10, Mab 2H7 andhumanized hMab 2F11-e7 to CSF1R fragment delD4/to CSF-1R-ECD was clearlybelow 1:50 (=0.02).

In a further experiment anti-CSF-1R antibodies 1.2.SM (ligand displacingCSF-1R antibody described in WO2009026303), CXIIG6 (ligand displacingCSF-1R antibody described in WO 2009/112245), the goat polyclonalanti-CSF-1R antibody ab10676 (abcam) were investigated. Anti-CSF-1Rantibody Mab3291 (R&D-Systems) was used as reference control. Anti-CCR5m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18.08.2004 at DSMZ) wasused as negative control.

TABLE 3b Binding of <CSF-1R> MAbs to human CSF-1R fragment delD4 andCSF-1R-ECD and ratio at 25° C., measured by SPR Binding Ratio of bindingof anti- Binding to CSF- CSF1R antibodies to to delD4 1R-ECD CSF1Rfragment delD4/ [RU] [RU] to CSF-1R-ECD MAB3291 1790 1222 1790/1222 =1.47 1.2.SM 469 704  469/704 = 0.67 CXIIG6 1983 1356 1983/1356 = 1.46ab10676 787 547  787/547 = 1.44 m<CCR5>Pz03.1C5 0 0 —

1.2.SM, CXIIG6, ab10676 and MAB3291 showed binding to CSF-1R-ECD and todel D4 (see FIGS. 3f and 3e ).

The binding ratio of 1.2.SM, CXIIG6, ab10676 and MAB3291 was highlyabove 1:50 (=0.02). Negative control antibody m<CCR5>Pz03.1C5 did notshow any binding (as expected).

Example 5 Growth Inhibition of NIH3T3-CSF-1R Recombinant Cells in 3DCulture Under Treatment with Anti-CSF-1R Monoclonal Antibodies(CellTiterGlo-Assay)

NIH 3T3 cells, retrovirally infected with either an expression vectorfor full-length wildtype CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301SY969F (SEQ ID NO: 63), were cultured in DMEM high glucose media (PAA,Pasching, Austria) supplemented with 2 mM L-glutamine, 2 mM sodiumpyruvate and non-essential amino acids and 10% fetal bovine serum(Sigma, Taufkirchen, Germany) on poly-HEMA(poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, Pa., USA))coated dishes to prevent adherence to the plastic surface. Cells areseeded in medium replacing serum with 5 ng/ml sodium selenite, 10 mg/mltransferrin, 400 μg/ml BSA and 0.05 mM 2-mercaptoethanol. When treatedwith 100 ng/ml hu CSF-1 (active 149 aa fragment of human CSF-1 (aa33-181 of SEQ ID NO: 86); Biomol, DE, Cat. No. 60530) wtCSF-1R(expressing cells form dense spheroids that grow three dimensionally, aproperty that is called anchorage independence. These spheroids resembleclosely the three dimensional architecture and organization of solidtumors in situ. Mutant CSF-1R recombinant cells are able to formspheroids independent of the CSF-1 ligand. Spheroid cultures wereincubated for 3 days in the presence of different concentrations ofantibody in order to determine an IC50 (concentration with 50 percentinhibition of cell viability). The CellTiterGlo assay was used to detectcell viability by measuring the ATP-content of the cells.

TABLE 4 Mutant wtCSF-1R IC50 CSF-1R IC50 CSF-1R Mab [μg/ml] [μg/ml] Mab2F11 1.1 8.0 Mab 2E10 0.49 4.9 Mab 2H7 0.31 5.3 Mab 1G10 0.29 14.2 SC2-4A5 10.0 10.0

Reference control Mab R&D-Systems 3291 did not show inhibition of mutantCSF-1R recombinant cell proliferation.

In a further experiment the anti-CSF-1R antibody according to theinvention hMab 2F11-e7 and the anti-CSF-1R antibodies 1.2.SM (liganddisplacing CSF-1R antibody described in WO 2009/026303), CXIIG6 (liganddisplacing CSF-1R antibody described in WO 2009/112245), the goatpolyclonal anti-CSF-1R antibody ab10676 (abcam), and SC 2-4A5 (SantaCruz Biotechnology, US— see also Sherr, C. J. et al., Blood 73 (1989)1786-1793) were investigated.

Spheroid cultures were incubated for 3 days in the presence of differentconcentrations of antibody in order to determine an IC30 (concentrationwith 30 percent inhibition of cell viability). Maximum concentration was20 μg/ml The CellTiterGlo assay was used to detect cell viability bymeasuring the ATP-content of the cells.

TABLE 5 Mutant wtCSF-1R IC30 CSF-1R IC30 CSF-1R Mab [μg/ml] [μg/ml] hMab2F11-e7 4.91 0.54 1.2.SM 1.19 >20 μg/ml (−19% inhibition at 20 μg/ ml =19% stimulation) CXIIG6 >20 μg/ml (21% >20 μg/ml (−36% inhibition at 20μg/ml) inhibition at 20 μg/ ml = 36% stimulation) ab10676 14.15 >20μg/ml (0% inhibition at 20 μg/ml) SC 2-4A5 16.62 2.56

Example 6 Growth Inhibition of BeWo Tumor Cells in 3D Culture UnderTreatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)

BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K media(Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2 mML-glutamine. 5×10⁴ cells/well were seeded in 96-well poly-HEMA(poly(2-hydroxyethylmethacrylate)) coated plates containing F12K mediumsupplemented with 0.5% FBS and 5% BSA. Concomitantly, 200 ng/ml huCSF-1(active 149 aa fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86)) and10 μg/ml of different anti-CSF-1R monoclonal antibodies were added andincubated for 6 days. The CellTiterGlo assay was used to detect cellviability by measuring the ATP-content of the cells in relative lightunits (RLU). When BeWo spheroid cultures were treated with differentanti-CSF-1R antibodies (10 μg/ml) inhibition of CSF-1 induced growth wasobserved. To calculate antibody-mediated inhibition the mean RLU valueof unstimulated BeWo cells was subtracted from all samples. Mean RLUvalue of CSF-1 stimulated cells was set arbitrarily to 100%. Mean RLUvalues of cells stimulated with CSF-1 and treated with anti-CSF-1Rantibodies were calculated in % of CSF-1 stimulated RLUs. The Table 6shows the calculated data of growth inhibition of BeWo tumor cells in 3Dculture under treatment with anti-CSF-1R monoclonal antibodies; FIGS. 2aand b depicts normalized mean RLU values.

TABLE 6 % inhibition 10 μg/ml CSF-1R Mab antibody concentration CSF-1only 0 Mab 2F11 70 Mab 2E10 102 Mab 2H7 103 Mab 1G10 99 SC 2-4A5 39

Example 7 Inhibition of Human Macrophage Differentiation Under Treatmentwith Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)

Human monocytes were isolated from peripheral blood using theRosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Tech.—Cat. No.15028). Enriched monocyte populations were seeded into 96 wellmicrotiterplates (2.5×10⁴ cells/well) in 100 μl RPMI 1640 (Gibco—Cat.No. 31870) supplemented with 10% FCS (GIBCO—Cat. No. 011-090014M), 4 mML-glutamine (GIBCO—Cat. No. 25030) and 1× PenStrep (Roche Cat. No. 1 074440) at 37° C. and 5% CO₂ in a humidified atmosphere. When 150 ng/mlhuCSF-1 was added to the medium, a clear differentiation into adherentmacrophages could be observed. This differentiation could be inhibitedby addition of anti-CSF-1R antibodies. Furthermore, the monocytesurvival is affected and could be analyzed by CellTiterGlo (CTG)analysis. From the concentration dependent inhibition of the survival ofmonocytes by antibody treatment, an IC₅₀ was calculated (see Table 7).

TABLE 7 IC50 CSF-1R Mab [μg/ml] Mab 2F11 0.08 Mab 2E10 0.06 Mab 2H7 0.03Mab 1G10 0.06 SC 2-4A5 0.36

In a separate test series humanized versions of Mab 2 F11, e.g. hMab2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, showed IC50 valuesof 0.07 μg/ml (hMab 2F11-c11), 0.07 μg/ml (hMab 2F11-d8), 0.04 μg/ml(hMab 2F11-e7) and 0.09 μg/ml (hMab 2F11-f12).

Example 8 Inhibition of Cynomolgous Macrophage Differentiation UnderTreatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)

Cynomolgous monocytes were isolated from peripheral blood using the CD14MicroBeads non-human primate kit (Miltenyi Biotec—Cat. No. 130-091-097)according to the manufacturers description. Enriched monocytepopulations were seeded into 96 well microtiterplates (1-3×10⁴cells/well) in 100 μl RPMI 1640 (Gibco—Cat. No. 31870) supplemented with10% FCS (GIBCO—Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO—Cat. No.25030) and 1× PenStrep (Roche Cat. No. 1 074 440) at 37° C. and 5% CO₂in a humidified atmosphere. When 150 ng/ml huCSF-1 was added to themedium, a clear differentiation into adherent macrophages could beobserved. This differentiation could be inhibited by addition ofanti-CSF-1R antibodies. Furthermore, the monocyte survival is affectedand could be analyzed by CellTiterGlo (CTG) analysis. The viability wasanalyzed at a concentration of 5 μg/ml antibody treatment (see Table 8).

TABLE 8 % inhibition (of survival) = CSF-1R Mab % survival (100% − %survival) Mab 2F11   4 * 96 Mab 2E10   17 ** 83 Mab 2H7 8 92 Mab 1G10 298 SC 2-4A5 31  69 * mean of four experiments (3 expts. using themurine, 1 expt. using the chimeric mAb) ** mean of two experiments usingthe murine mAb only

Example 9 Inhibition of Human M1 and M2 Macrophage Differentiation UnderTreatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo-Assay)

Human monocytes were isolated from peripheral blood using theRosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Tech.—Cat. No.15028). Enriched monocyte populations were seeded into 96 wellmicrotiterplates (2.5×10⁴ cells/well) in 100 μl RPMI 1640 (Gibco—Cat.No. 31870) supplemented with 10% FCS (GIBCO—Cat. No. 011-090014M), 4 mML-glutamine (GIBCO—Cat. No. 25030) and 1× PenStrep (Roche Cat. No. 1 074440) at 37° C. and 5% CO₂ in a humidified atmosphere. When 100 ng/mlhuCSF-1 was added for 6 days to the medium, a clear differentiation intoadherent, M2 macrophages with elongated morphology could be observed.When 100 ng/ml huGM-CSF was added to the medium for 6 days, a cleardifferentiation into adherent, M1 macrophages with round morphologycould be observed. This differentiation was associated with theexpression of certain markers such as CD163 for M2 macrophages and CD80or high MHC class II for M1 macrophages as assessed by flow cytometry.Cells were washed with PBS and, if adherent, detached using a 5 mM EDTAsolution in PBS (20 min at 37° C.). Cells were then well resuspended,washed with staining buffer (5% FCS in PBS) and centrifuged at 300×g for5 min. Pellets were resuspended in 1 ml staining buffer and cellscounted in a Neubauer chamber. Approximately 1×10e5 cells weretransferred in each FACS tube, centrifuged at 300×g for 5 min andresuspended in staining buffer. Fcγ receptors were blocked by incubationwith 1 μg human IgG/2.5×10e4 cells (JIR Cat. No. 009-000-003) instaining buffer for 20 min on ice. Cells were then mixed with 1.5 μlantibody/2.5×10e4 cells for CD80 and CD163 detection whereas 5 μlantibody/2.5×10e4 cells for MHC class II detection was used: PE labeledmouse anti human CD163 (BD Bioscience Cat. No. 556018), PE labeled mouseanti human CD80 (BD Bioscience Cat. No. 557227) and Alexa 647 labeledmouse anti human MHC class II (Dako—Cat. No. M0775). The Alexa 647 labelwas conjugated to the antibody by using the Zenon Alexa 647 mouse IgGlabeling kit (Invitrogen Cat. No. Z25008) After a 1-hour incubation onice cells were washed twice with staining buffer, resuspended andmeasured at a FACS Canto II.

Exclusively M2 macrophage differentiation which is characterized by theexpression of CD163, absence of CD80 and low MHC class II expressioncould be inhibited by addition of humanized anti-CSF-1R antibody hMab2F11-e7. Furthermore, the M2 but not M1 macrophage survival is affectedand could be analyzed by CellTiterGlo (CTG) analysis. Concentrationdependent inhibition of the survival of macrophages by antibodytreatment for 7 days is depicted in FIG. 5a . Expression of M1 and M2macrophage markers assessed by flow cytometry is shown in FIG. 5 b.

Example 10 Determination of the Binding Affinity of Anti-CSF-1RAntibodies to Human CSF-1R Instrument: BIACORE® A100 Chip: CMS (BiacoreBR-1006-68)

Coupling: amine coupling

Buffer: PBS (Biacore BR-1006-72), pH 7.4, 35° C.

For affinity measurements 36 μg/ml anti mouse Fcγ antibodies (from goat,Jackson Immuno Research JIR115-005-071) have been coupled to the chipsurface for capturing the antibodies against CSF-1R. Human CSF-1RExtracellular Domain (CSF-1R-ECD) (comprising the extracellularsubdomains D1-D5) (SEQ ID NO: 64) (R&D-Systems 329-MR or subclonedpCMV-presS-HisAvitag-hCSF-1R-ECD) was added in various concentrations insolution. Association was measured by an CSF-1R-injection of 1.5 minutesat 35° C.; dissociation was measured by washing the chip surface withbuffer for 10 minutes at 35° C. For calculation of kinetic parametersthe Langmuir 1:1 model was used.

TABLE 9 Affinity data measured by SPR KD ka kd t½ CSF-1R Mab (nM) (1/Ms)(1/s) (min) Mab 2F11 0.29 1.77E+05 5.18E−05 223 Mab 2E10 0.2 1.52E+052.97E−05 389 Mab 2H7 0.21 1.47E+05 3.12E−05 370 Mab 1G10 0.36 1.75E+056.28E−05 184

In a separate biacore binding assay using the CSF-1R ECD (data notshown) some competition of the antibodies Mab 2F11 and Mab 2E10 with theantibody Ab SC-2-4A5 was shown. However Mab 2F11/Mab 2E10 do not bind tothe human CSF-1R fragment delD4, whereas Ab SC-2-4A5 binds to this delD4fragment (see Example 4 and FIG. 3a ). Thus the binding region of Mab2F11/Mab 2E10 is clearly distinct from the binding region of AbSC-2-4A5, but probably located in a vicinity area. In such competitionassay both antibodies Mab 2F11 and Mab 2E10 did not compete with Mab3291from R&D-Systems (data not shown).

Example 11 Determination of the Binding of Anti-CSF-1R Antibodies toHuman CSF-1R Fragment D1-D3 Instrument: Biacore T100 (GE Healthcare)

-   -   Software: T100 Control, Version 1.1.11        -   B3000 Evaluation, Version 4.01        -   Scrubber, Version 2.0a

Assayformat Chip:CMS-Chip

Antibodies against CSF-1R were captured via amine coupled capturemolecules. Using the single cycle kinetics five increasingconcentrations of human CSF-1R fragment D1-D3 (SEQ ID NO: 66) wereinjected. Human CSF-1R fragment D1-D3 was subcloned intopCMV-presS-HisAvitag expression vector.

Anti CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US; Sherr, C. J. et al.,Blood 73 (1989) 1786-1793) which inhibits the ligand-receptorinteraction, and Mab 3291 (R&D-Systems) were used as reference controls.

Capture molecules: Anti mouse Fcγ antibodies (from goat, Jackson ImmunoResearch JIR115-005-071) for antibodies according to the invention andthe R&D-Systems control Mab 3291 and Anti rat Fcγ antibodies (from goat,Jackson Immuno Reasearch JIR112-005-071) for the reference control antiCSF-1R SC 2-4A5.

Amine Coupling of Capture Molecules

Standard amine coupling according to the manufacturer's instructions:running buffer: HBS-N buffer, activation by mixture of EDC/NHS, aim forligand density of 2000 RU; the capture-Abs were diluted in couplingbuffer NaAc, pH 4.5, c=10 mg/mL; finally remaining activated carboxylgroups were blocked by injection of 1 M Ethanolamin.

Kinetic Characterization of Human CSF-1R Fragments D1-D3 Binding to MAbs<CSF-1R> at 37° C. Running Buffer: PBS (Biacore BR-1006-72)

Capturing of Mabs <CSF-1R> on flow cells 2 to 4: Flow 20 μl/min, contacttime 90 seconds, c(Abs<CSF-1R>)=50 nM, diluted with running buffer+1mg/mL BSA;

Analyte Sample:

Single Cycle Kinetics was measured at a flow rate of 30 μL/min by fiveconsecutive injections of the analyte with concentrations, c=7.8, 31.25,125 500 and 2000 nM, without regeneration. Each injection was 30 secondslong and followed by a dissociation phase of 120 Seconds for the firstfour injections, and finally 1200 seconds for the highest concentration(=last injection).

Final regeneration was performed after each cycle using 10 mM Glycin pH1.5 (Biacore BR-1003-54), contact time 60 seconds, flow rate 30 μL/min.

Kinetic parameters were calculated by using the usual double referencing(control reference: binding of analyte to capture molecule; Flow Cell:subdomain CSF-1R concentration “0” as Blank) and calculation with model‘titration kinetics 1:1 binding with draft’.

TABLE 10 Affinity data for binding of human CSF-1R fragment D1-D3measured by SPR Sub KD ka kd t½ CSF-1R Mab domain (nM) (1/Ms) (1/s)(min) Mab 2F11 D1-D3 no binding Mab 2E10 D1-D3 no binding Mab 2H7 D1-D3not determined Mab 1G10 D1-D3 no binding SC-2-4A5 D1-D3 no bindingR&D-Systems D1-D3 5.4 2.2E+5 1.2E−3 9.6 3291

The antibodies Mab 2F11, Mab 2E10 and Mab 1G10 showed no binding tohuman CSF-1R fragment D1-D3.

Also reference control-Ab SC-2-4A5 did not bind to human CSF-1R fragmentD1-D3.

The reference control Mab R&D-Systems 3291 showed binding to the humanCSF-1R fragment D1-D3.

Example 12 CSF-1 Level Increase During CSF-1R Inhibition in CynomolgusMonkey

Serum CSF-1 levels provide a pharmacodynamic marker of CSF-1Rneutralizing activity of anti-human CSF-1R dimerization inhibitor hMab2F11-e7. One male and one female cynomolgus monkey per dosage group (1and 10 mg/kg) were intravenously administered anti-CSF1R antibody hMab2F11-e7. Blood samples for analysis of CSF-1 levels were collected 1week before treatment (pre-dose), 2, 24, 48, 72, 96, 168 hours post-doseand weekly for two additional weeks. CSF-1 levels were determined usinga commercially available ELISA kit (Quantikine® human M-CSF) accordingto the manufacturer's instructions (R&D Systems, UK). Monkey CSF-1 levelwere determined by comparison with CSF-1 standard curve samples providedin the kit.

Administration of hMab 2F11-e7 induced a dramatic increase in CSF-1 by˜1000-fold, which depending on the dose administered lasted for 48 hr (1mg/kg) or 15 days (10 mg/kg). Hence, a dimerization inhibitor for CSF-1Roffers the advantage to not directly compete with the dramaticallyupregulated ligand for binding to the receptor in contrast to a liganddisplacing antibody. (Results are shown in FIG. 4)

Example 13 Inhibition of Tumor Growth Under Treatment with Anti-CSF-1RMonoclonal Antibody in Combination with Chemotherapy or CancerImmunotherapy in Subcutaneous Syngeneic MC38 Colon Carcinoma Models

Cells of the murine colorectal adenocarcinoma cell line MC-38 (obtainedfrom Beckman Research Institute of the City of Hope, California, USA)were cultured in Dulbecco's Modified Eagle Medium (DMEM, PAN Biotech)supplemented with 10% FCS and 2 mM L-glutamine at 37° C. in a watersaturated atmosphere at 5% CO2. At the day of inoculation, MC38 tumorcells were harvested with PBS from culture flasks and transferred intoculture medium, centrifuged, washed once and re-suspended in PBS. Forinjection of cells, the final titer was adjusted to 1×107 cells/ml.Subsequently 100 μl of this suspension (1×106 cells) were inoculatedsubcutaneously into 7-9 weeks old female C57BL/6N mice (obtained fromCharles River, Sulzfeld, Germany). Treatment with control antibody(MOPC-21; Bio X Cell, West Lebanon), anti-murine CSF-1R mAb <mouseCSF1R> antibody at a weekly dose of 30 mg/kg i.p. alone or incombination the TLR9 agonist CpG ODN 1826 (ODN 1826, class B CpG ODN,VacciGrade, InvivoGen, 100 μg peritumoral, 1×). Tumor volume wasmeasured twice a week and animal weights were monitored in parallel.

In a separate study with comparable set-up, primary tumors fromindicated treatment groups were excised, weighed and subjected to FACSanalysis. Primary tumor material was collected between study day 20-25as indicated. To obtain single cell suspensions amenable for flowcytometry analysis the tumors were minced by using the Mcllwain tissuechopper. Subsequently, the tumor pieces were resuspended in RPMI mediasupplemented with collagenase I, dispase II and DNAse I, incubated at37° C. and cell suspension were passed through a mash. CD45 positivecells were enriched by magnetic cell separation according to themanufacturer's instructions (Miltenyi). Briefly cells were labeled withanti-mouse CD45 conjugated with APC (BD, Cat. No 559864) and separatedwith anti APC microbeads. To analyse CD8+ T cells these CD45 positivecells were stained with 0.2 μg/ml DAPI (Roche, Cat. No 10236276001 andPE conjugated CD8 antibody (eBioscience Cat. No. 12-0081-83) or PEconjugated CD4 antibody (eBioscience, Cat. No. 2-0041-83). Acquisitionof data was performed with FACS Canto II and subsequently analysed withFlowJo software. Only viable cells (gated on DAPI-negative cells) wereanalysed to exclude cell debris and dead cells.

Monotherapy with <mouse CSF1R> antibody inhibited primary tumor growthwhen compared to control antibody treatment (TGI: 67%, TCR: 0.38 CI:0.15-0.64). Also TLR9 agonist (CpG ODN 1826) monotherapy had an effecton MC38 primary tumor growth (TGI: 74%, TCR: 0.28 CI: 0.10-0.50).Addition of <mouse CSF1R> antibody to TLR9 agonist therapy led to aclearly superior anti-tumor efficacy compared to TLR9 agonist treatmentalone (TGI: 95%, TCR: 0.08 CI: −0.11-0.28). (see table 11).

TABLE 11 Anti tumor Efficacy of <mouse CSF1R> antibody alone and incombination with TLR9 agonist CpG in the MC38 mouse CRC in vivo modelMedian time to TGI TCR progression Group (day 24) (day 24) TV > 700 mm3Control (Mouse IgG1) — — 21 <mouse CSF1R> 67% 0.38 24 antibody TLR9agonist (CpG) 73% 0.28 24 <mouse CSF1R> 95% 0.08 46 antibody/TLR9agonist (CpG)

Evaluation on Tumor Progression

Additionally to the assessment of median tumor volume after 2 weeks oftreatment the progression of individual tumors in the study was followeduntil progression ≧700 mm3 (FIG. 1 (CpG is TLR9 agonist CpG ODN 1826)and Table 11)

Median time to progression ≧700 mm3 was 21 days for IgG controltreatment group. Slight improvement of median progression time wasachieved by treatment with <CSF1R> antibody monotherapy (24 days).

Monotherapy with TLR9 agonist resulted in a median time to progressionof 24 days. Addition of TLR9 agonist (CpG) to anti-CSF-1R antibodytherapy resulted in a statistically significant more than additiveimprovement of median time to progression (46 days) compared toanti-CSF-1R antibody monotherapy or TLR9 agonist monotherapy.

1. A composition comprising an antibody which binds to human CSF-1R anda Toll-like receptor 9 (TLR9) agonist.
 2. The composition of claim 1,wherein the antibody does not bind to human CSF-1R fragment delD4 (SEQID NO: 65).
 3. The composition of claim 1, wherein the antibodycomprises a) a heavy chain variable domain comprising SEQ ID NO:7 and alight chain variable domain comprising SEQ ID NO:8, b) a heavy chainvariable domain comprising SEQ ID NO:15 and a light chain variabledomain comprising SEQ ID NO:16; c) a heavy chain variable domaincomprising SEQ ID NO:75 and a light chain variable domain comprising SEQID NO:76; d) a heavy chain variable domain comprising SEQ ID NO:83 and alight chain variable domain comprising SEQ ID NO:84; e) a heavy chainvariable domain comprising SEQ ID NO:23 and a light chain variabledomain comprising SEQ ID NO:24, or f) a heavy chain variable domaincomprising SEQ ID NO:31 and a light chain variable domain comprising SEQID NO:32, or g) a heavy chain variable domain comprising SEQ ID NO:39and a light chain variable domain comprising SEQ ID NO:40, or h) a heavychain variable domain comprising SEQ ID NO:47 and a light chain variabledomain comprising SEQ ID NO:48, or i) a heavy chain variable domaincomprising SEQ ID NO:55 and a light chain variable domain comprising SEQID NO:56.
 4. The antibody of claim 1, wherein the antibody comprises a)a heavy chain variable domain comprising a CDR3 region of SEQ ID NO: 1,a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ ID NO:3, and alight chain variable domain comprising a CDR3 region of SEQ ID NO: 4, aCDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6, or b) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO: 9, aCDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and alight chain variable domain comprising a CDR3 region of SEQ ID NO:12, aCDR2 region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO: 14, or c)a heavy chain variable domain comprising a CDR3 region of SEQ ID NO: 17,a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO:19, and alight chain variable domain comprising a CDR3 region of SEQ ID NO: 20, aCDR2 region of SEQ ID NO:21, and a CDR1 region of SEQ ID NO:22, or d) aheavy chain variable domain comprising a CDR3 region of SEQ ID NO: 25, aCDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and alight chain variable domain comprising a CDR3 region of SEQ ID NO:28, aCDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or e)a heavy chain variable domain comprising a CDR3 region of SEQ ID NO: 33,a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, anda light chain variable domain comprising a CDR3 region of SEQ ID NO:36,a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, orf) a heavy chain variable domain comprising a CDR3 region of SEQ IDNO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ IDNO:43, and a light chain variable domain comprising a CDR3 region of SEQID NO: 44, a CDR2 region of SEQ ID NO:45, and a CDR1 region of SEQ IDNO:46, or g) a heavy chain variable domain comprising a CDR3 region ofSEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQID NO: 51, and a light chain variable domain comprising a CDR3 region ofSEQ ID NO:52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQID NO: 54; or h) a heavy chain variable domain comprising a CDR3 regionof SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region ofSEQ ID NO:71, and a light chain variable domain comprising a CDR3 regionof SEQ ID NO: 72, a CDR2 region of SEQ ID NO:73, and a CDR1 region ofSEQ ID NO:74, or i) a heavy chain variable domain comprising a CDR3region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1region of SEQ ID NO: 79, and a light chain variable domain comprising aCDR3 region of SEQ ID NO:80, a CDR2 region of SEQ ID NO: 81, and a CDR1region of SEQ ID NO:
 82. 5. (canceled)
 6. (canceled)
 7. (canceled) 8.(canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled) 17.(canceled)